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Proteasome Inhibitors as anticancer drugs
Abstract
The targeted degradation of key regulatory proteins is an essential element of cell cycle control. The proteasome plays a central role in the degradation of such proteins and has therefore become an important therapeutic target for diseases involving cell proliferation, notably cancer. This review summarizes numerous studies demonstrating that proteasome inhibition induces apoptosis and sensitizes cancer cells to traditional tumoricidal agents both and The potent and selective proteasome inhibitor, PS-341, is particularly promising from a therapeutic perspective, and it is the only such inhibitor that has progressed to clinical trials. Preliminary data indicate that the drug is well tolerated by patients with cancer, and further trials are underway to assess the safety and efficacy of proteasome inhibition in hematologic and solid tumors, both as a monotherapy and in combination with other chemotherapeutics.
... Bortezomib was first synthesized in 1995 and subsequently approved in 2003 as a preferential treatment for multiple myeloma 22 . Bortezomib has been reported to show a potent anticancer activity, both in vitro and in vivo, also in other cancer cell lines, like prostate cancer, pancreatic cancer, renal cell carcinoma and squamous cell carcinoma [23][24][25][26] . In addition, several clinical trials evaluated the effect of Bortezomib in combination with chemotherapy or other targeted therapies in HNC 27-34 . ...
... The purpose of proteasome inhibitors is to block the turnover of the intracellular proteome, bringing about the formation of a toxic environment and the subsequent need to activate apoptotic processes 42,43 . In this regard, Bortezomib is a reversible inhibitor of the chymotrypsin-like activity of the 20S proteasome 14,20,21 and it has been reported to possess potent anticancer activity, both in vitro and in vivo [23][24][25][26]44,45 . ...
Head and neck cancer (HNC) has frequently an aggressive course for the development of resistance to standard chemotherapy. Thus, the use of innovative therapeutic drugs is being assessed. Bortezomib is a proteasome inhibitor with anticancer effects. In vitro antitumoral activity of Bortezomib was investigated employing human tongue (SCC-15, CAL-27), pharynx (FaDu), salivary gland (A-253) cancer cell lines and a murine cell line (SALTO-5) originated from a salivary gland adenocarcinoma arising in BALB-neuT male mice transgenic for the oncogene neu. Bortezomib inhibited cell proliferation, triggered apoptosis, modulated the expression and activation of pro-survival signaling transduction pathways proteins activated by ErbB receptors and inhibited proteasome activity in vitro. Intraperitoneal administration of Bortezomib delayed tumor growth of SALTO-5 cells transplanted in BALB-neuT mice, protracted mice survival and adjusted tumor microenvironment by increasing tumor-infiltrating immune cells (CD4⁺ and CD8⁺ T cells, B lymphocytes, macrophages, and Natural Killer cells) and by decreasing vessels density. In addition, Bortezomib modified the expression of proteasome structural subunits in transplanted SALTO-5 cells. Our findings further support the use of Bortezomib for the treatment of HNC and reveal its ineffectiveness in counteracting the activation of deregulated specific signaling pathways in HNC cell lines when resistance to proteasome inhibition is developed.
... Bortezomib was rst synthesized in 1995 and subsequently approved in 2003 as a preferential treatment of multiple myeloma 22 . Bortezomib has been reported to show a potent anticancer activity, both in vitro and in vivo, also in other cancer cell lines, like prostate cancer, pancreatic cancer, renal cell carcinoma and squamous cell carcinoma [23][24][25][26] . In addition, several clinical trials evaluated the effect of Bortezomib in combination with chemotherapy or other targeted therapies in HNC [27][28][29][30][31][32][33][34] . ...
... It is a speci c and reversible inhibitor of the chymotrypsin-like activity of the 20S proteasome14,20,21 . Bortezomib has been reported to possess potent anticancer activity, both in vitro and in vivo, in prostate cancer, pancreatic cancer, renal cell carcinoma, and squamous cell carcinoma[23][24][25][26]44,45 .Preclinical studies have shown that Bortezomib, when used alone, inhibited cells growth, induced apoptosis, autophagy, and modulation of individual signaling transduction pathways in HNSCC (Head and neck squamous cell carcinoma) cells in vitro 46-56 . However, none of these studies have analyzed the simultaneous effects of Bortezomib on proteasome, apoptosis, autophagy and signaling transduction pathways involved in cellular transformation in HNC cell lines. ...
Head and neck cancer (HNC) has frequently an aggressive course for the development of resistance to standard chemotherapy. Thus, the use of innovative therapeutic drugs is being assessed. Bortezomib is a proteasome inhibitor with strong in vitro and in vivo anticancer effects. In vitro antitumoral activity of Bortezomib was investigated employing human pharynx (FaDu), tongue (SCC-15, CAL-27), salivary gland (A-253) cancer cell lines and a murine cell line (SALTO-5) originated from a salivary gland adenocarcinoma arising in BALB- neu T male mice transgenic for the oncogene neu. Bortezomib in vivo effects in BALB- neu T mice transplanted with murine SALTO-5 cells were also examined. Bortezomib inhibited cells proliferation, triggered apoptosis, modulated the expression and activation of pro-survival signal transduction pathways proteins activated by ErbB receptors and inhibited proteasome activity in vitro . Furthermore, intraperitoneal administration of Bortezomib delayed tumor growth of SALTO-5 cells transplanted in BALB- neu T mice and protracted mice survival. Our findings further support the use of Bortezomib for the treatment of HNC and reveal its ineffectiveness in counteracting the activation of deregulated specific signaling pathways in HNC cell lines when resistance to proteasome inhibition is developed.
... Bortezomib (BZ; PS-341) is a boronic acid dipeptide that inhibits 26S proteasome activity (9). The antitumor effects of BZ have been extensively studied in multiple myeloma (MM) cells. ...
... BZ is a highly selective, reversible inhibitor of the 26S proteasome that is recommended for single-agent use in the treatment of patients with multiple myeloma (9)(10)(11)(12)(13). BZ has been known to have a variety of mechanisms influencing the physiology of cells, which indicates that exposure to BZ has the exposure to BZ results in the stabilization of p21, p27 and p53, as well as the pro-apoptotic Bid and Bax proteins, caveolin-1 and inhibitor κB-α, which prevents the activation of NF-κB-induced cell survival pathways. ...
The progression of primary prostate cancer (PC) is dependent on the androgen receptor (AR) and prostate-specific membrane antigen (PSMA). Furthermore, the growth of PC cells is terminated with the downregulation of both AR and PSMA. In our preliminary experiments, it was also found that bortezomib (BZ; PS-341) that inhibits 26S proteasome activity, acts as a downregulator of both PSMA and AR. In addition to evaluating the effects of BZ on protein expression, the present study evaluated and compared the anticancer effects of BZ on the growth of cells treated with BZ, docetaxel (DOC), or a combination of both. Western blot analysis was used to examine the expression levels of AR and PSMA. The knockdown effect of small interfering RNA (siRNA) and the drugs on the expression of either AR or PSMA was also evaluated. An MTT assay was performed in order to evaluate the inhibitory effects of the drugs on PC cells. The cell cycles were analyzed, and apoptotic cells were detected. The downregulation of AR and PSMA was observed using siRNA specific to AR or PSMA, and the inhibition of PSMA, as well as that of AR severely suppressed the growth of PC cells. The inhibitory effect of BZ alone on PSMA expression was similar to that of both AR- and PSMA-specific siRNA, and this drug also induced the downregulation of AR and PSMA in PC cells. This phenomenon was confirmed even in cells transfected to overexpress PSMA. The apoptosis-promoting effect of BZ on the cells was similar to that observed with BZ plus DOC, and more potent than that of DOC alone. BZ had the same inhibitory effect on the expression of AR and PSMA as did siRNA specific to AR or PSMA. On the whole, the findings of this study indicate that BZ may prove to be a promising chemotherapeutic agent and may be used as a molecularly targeted drug in the treatment of PC.
... Because there are approximately 600 ubiquitin E3 ligases in humans, each with a limited number of substrates and several with biochemical or genetic links to various cancers, it seemed reasonable that at least a few ligases would be ideal anticancer targets. Since these early days of limited expectations for proteasome inhibitors and great hopes for E3 ligase inhibitors, the relatively " unselective " proteasome inhibitors have undergone extensive clinical evaluation, resulting in two approvals so far by the USFDA [3, 4], while their more " selective " counterparts, inhibitors of ubiquitin E3 ligases, have struggled in development by comparison ...
... Bortezomib. The first (and until 2012 the only) clinically approved proteasome inhibitor, bortezomib (VELCADE ® ), a synthetic peptidyl boronate developed by Millennium Pharmaceuticals (Takeda) (Fig. 3) [15], was initially approved as an injectable treatment for refractory relapsed multiple myeloma and, shortly afterward, for first line treatment of multiple myeloma and treatment of relapsed mantle cell lymphoma [4, 16, 17]. Bortezomib binds reversibly to the 5 subunit of the 20S core (inhibits the threonine protease activity of the Chymotrypsin-like subunit, the rate limiting step of proteolysis, by forming a complex between its boronic acid group and the threonine hydroxyl group at the catalytic site [6, 18]. ...
Molecular oncology has the potential to revolutionize cancer treatment owing to its focus on discrete, cancer-selective targets, as evident in the recent success of kinase inhibitors and antibody-based therapies. Because of the heterogeneous nature of cancer, however, not every tumor type can be addressed with an appropriately selective therapy and some respond best to drug combinations that include classical “toxic” agents. The ubiquitin-proteasome pathway, recently harnessed for cancer treatment with the clinical use of “toxic” proteasome inhibitors bortezomib and carfilzomib, affords targets that intuitively are highly selective, exemplified by inhibitors of E3 ligases, the ubiquitin-conjugating enzymes, as well as those that are intuitively nonselective, exemplified by the proteasomal proteases. In the last two decades, anticancer drug development based on these two target classes has proceeded in parallel, with the early results suggesting that the nonselective proteasome is the better target. Lately, however, it has become clear that (1) the “nonselective” proteasome target may be addressed in selective ways and (2) a clearer understanding of the E3 ligase reaction can lead to the design or discovery of efficacious inhibitors. Evidence supporting these notions and implications for cancer treatment going forward will be discussed.
... The ubiquitin-proteasome system (UPS) catalyzes the degradation of ubiquitinated intracellular proteins123. Over the past decades, the role of UPS has proven to be a crucial regulator of cell proliferation and survival by controlling cellular levels of key proteins in these processes [4,5]. Polyubiquitinated proteins are transported to the 26S proteasome for degradation where the 19S cap recognizes, denatures and directs the targeted protein to the catalytic 20S core of the proteasome. ...
... Inhibition of the UPS results in perturbation of intracellular protein homeostasis by accumulation of the poly-ubiquitinated proteins, subsequently inducing cellular stress and apoptosis. Interestingly, UPS inhibition appeared to preferentially affect malignant cells reflecting a higher dependence of tumor cells on the UPS [5]. This finding prompted the development of proteasome inhibitors (PIs). ...
... Moreover, some compounds showed a higher inhibition of the chymotryptic activity in the lysates of solid cancer cell lines than in the ones of hematological cancers, e.g., compounds 1 (CT-L: 54% in Jurkat, 63% in K562, 62% in HT-29, 90% in HCT116) and 5 (CT-L: 64% in Jurkat, 81% in HT-29, 67% in HCT116). However, despite these results, it is important to consider that, in vivo, this pattern might change because, usually, proteasome inhibitors have a limited success in clinical trials for solid tumors largely due to poor tissue penetration as well as a rapid clearance from the bloodstream [56,57]. ...
Cancer is a complex multifactorial disease whose pathophysiology involves multiple metabolic pathways, including the ubiquitin–proteasome system, for which several proteasome inhibitors have already been approved for clinical use. However, the resistance to existing therapies and the occurrence of severe adverse effects is still a concern. The purpose of this study was the discovery of novel scaffolds of proteasome inhibitors with anticancer activity, aiming to overcome the limitations of the existing proteasome inhibitors. Thus, a structure-based virtual screening protocol was developed using the structure of the human 20S proteasome, and 246 compounds from virtual databases were selected for in vitro evaluation, namely proteasome inhibition assays and cell viability assays. Compound 4 (JHG58) was shortlisted as the best hit compound based on its potential in terms of proteasome inhibitory activity and its ability to induce cell death (both with IC50 values in the low micromolar range). Molecular docking studies revealed that compound 4 interacts with key residues, namely with the catalytic Thr1, Ala20, Thr21, Lys33, and Asp125 at the chymotrypsin-like catalytic active site. The hit compound is a good candidate for additional optimization through a hit-to-lead campaign.
... Proteotoxic stress is a common condition in malignant cells [13]. The constant challenge of keeping the negative consequences of proteotoxic stress in check increases the sensitivity of cancer cells to drugs that interfere with protective mechanisms aimed at restoring proteostasis, such as the UPS and autophagy [14,15]. The increased load of misfolded proteins in malignant cells as well as their altered metabolic state generates a therapeutic window at which limiting the activity of these proteolytic systems has lethal consequences for cancer cells without causing substantial collateral damage to healthy, untransformed cells. ...
The ubiquitin-proteasome system (UPS) and macroautophagy/autophagy are the main proteolytic systems in eukaryotic cells for preserving protein homeostasis, i.e., proteostasis. By facilitating the timely destruction of aberrant proteins, these complementary pathways keep the intracellular environment free of inherently toxic protein aggregates. Chemical interference with the UPS or autophagy has emerged as a viable strategy for therapeutically targeting malignant cells which, owing to their hyperactive state, heavily rely on the sanitizing activity of these proteolytic systems. Here, we report on the discovery of CBK79, a novel compound that impairs both protein degradation by the UPS and autophagy. While CBK79 was identified in a high-content screen for drug-like molecules that inhibit the UPS, subsequent analysis revealed that this compound also compromises autophagic degradation of long-lived proteins. We show that CBK79 induces non-canonical lipidation of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 beta) that requires ATG16L1 but is independent of the ULK1 (unc-51 like autophagy activating kinase 1) and class III phosphatidylinositol 3-kinase (PtdIns3K) complexes. Thermal preconditioning of cells prevented CBK79-induced UPS impairment but failed to restore autophagy, indicating that activation of stress responses does not allow cells to bypass the inhibitory effect of CBK79 on autophagy. The identification of a small molecule that simultaneously impairs the two main proteolytic systems for protein quality control provides a starting point for the development of a novel class of proteostasis-targeting drugs.
... A proteasome is a multi-subunit holoenzyme responsible for proteolysis of most intracellular proteins in eukaryotic cells, and they provide prosurvival effects through cell cycle progression, endoplasmic reticulum (ER) homeostasis, and NF-kB signaling (Thibaudeau and Smith 2019;Adams 2004). Inhibition of proteasome catalytic sites in neoplastic PCs results in apoptosis (Adams 2002a(Adams , b, 2004. In fact, the proteasome inhibitor bortezomib (BTZ) has been approved for the treatment of patients with multiple myeloma (MM) and mantle cell lymphoma (MCL) (Thibaudeau and Smith 2019;Adams 2004;Richardson et al. 2005). ...
Autoimmune diseases (ADs) are conditions in which the immune system cannot distinguish self from non-self and, as a result, tissue injury occurs primarily due to the action of various inflammatory mediators. Different immunosuppressive agents are used for the treatment of patients with ADs, but some clinical cases develop resistance to currently available therapies. The proteasome inhibitor bortezomib (BTZ) is an approved agent for first-line therapy of people with multiple myeloma. BTZ has been shown to improve the symptoms of different ADs in animal models and ameliorated symptoms in patients with systemic lupus erythematous, rheumatoid arthritis, myasthenia gravis, neuromyelitis optica spectrum disorder, Chronic inflammatory demyelinating polyneuropathy, and autoimmune hematologic diseases that were nonresponsive to conventional therapies. Proteasome inhibition provides a potent strategy for treating ADs. BTZ represents a proteasome inhibitor that can potentially be used to treat AD patients resistant to conventional therapies.
... The ubiquitin-proteasome pathway is the most important intracellular proteolytic system and its function is crucial to cellular homeostasis [1,2]. Target proteins are often degraded by the 26S proteasome after labeling with the 76 amino acid ubiquitin molecule. ...
The ubiquitin–proteasome pathway is an important protein quality control system involved in intracellular homeostasis. To achieve intracellular homeostasis, proteins that are misfolded as a result of translational errors or genetic mutations must be eliminated by the ubiquitin–proteasome pathway. In our previous publications, we determined that 4T1 breast and B16F10 melanoma cancer cells have differential levels of resistance to proteasome inhibitors. Again, in the previous studies, we reported that 4T1 cell cultures, despite being p53-mutant, underwent apoptosis as a result of bortezomib treatment. The first goal of this study was to verify the resistance levels of parental and resistant PC3 prostate cancer cells to bortezomib using WST-1 test. As a result of treatment with different bortezomib concentrations for 48 h, the IC50 value of the parental cells was determined as 32.8 nM and that of the resistant cells was determined as 346 nM. This result showed that the resistant cells were at least 10.5 times more resistant. In addition, to determine whether the resistance gained was reversible or not, the cells were passaged in a medium without bortezomib for one month. The IC50 value determination by WST-1 test showed that the resistant PC3 cells gained an irreversible bortezomib resistance phenotype. The results of the 3D spheroid experiment showed that the 3D spheroid diameter of resistant cells was significantly higher than that of the parental cells. The studies conducted with Western blot showed that ERK1 MAPK T202 phosphorylation and the conversion of autophagy marker LC3-I to LC3-II were significantly increased in parental cells as compared to the resistant cells. Finally, the results showed that while both maternal embryonic leucine zipper kinase (MELK) inhibitor OTSSP167 and Ca2+ chelator BAPTA-AM (also an inhibitor of the expression of antiapoptotic protein GRP78) are promising agents for cancer cells resistant to the proteasome inhibitors, CDK2 inhibitor CVT-313 was found ineffective in both parental and the resistant cells.
... The antitumor activities of several proteasome inhibitors [e.g., bortezomib (BZM) and carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132)] have been validated in clinical or preclinical studies in multiple myeloma and other cancers [8,9]. BZM, which targets 26S proteasome activity, is a first-in class proteasome inhibitor drug approved by the FDA for the treatment of multiple myeloma (MM), relapsed/refractory MM, and mantle cell lymphoma [10]. MG132, an inhibitor of the 26S proteasome, exhibits antitumor activity by inducing apoptosis in tumor cells through various mechanisms [11]. ...
Both mitoxantrone (MTX) and proteasome inhibitors efficiently trigger immunogenic cell death (ICD) in cancer cells. However, whether the combination of MTX and proteasome inhibitors can synergistically enhance ICD remains unknown. In this study, we showed that the proteasome inhibitors bortezomib (BZM) and carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132) impaired MTX-induced ICD in prostate cancer cells, as measured using ICD biomarkers and dendritic cell activation in vitro. Mice vaccinated with RM-1 mouse prostate cancer cell line treated with BZM or MG132 in combination with MTX showed enhanced tumor growth, and shortened tumor-free, and worse overall survival compared with those treated with MTX alone. In conclusion, we demonstrated that proteasome inhibitors (BZM or MG132) attenuated MTX-induced ICD, suggesting that proteasome activation was required for MTX-induced ICD.
... As proteasome plays an essential role in regulation of important physiological processes such as the cell cycle and apoptosis, proteasome inhibitors have been extensively studied in the cancer field [15,16]. Many therapeutic strategies for treating different types of cancers utilize the ability of the proteasome inhibitors to induce cancer cell apoptosis [13,17]. PS-341 (also called Bortezomib) is one such proteasome inhibitor now approved by the FDA for treatment of multiple myeloma, and many other proteasome inhibitors are at various stages of clinical development [18,19]. ...
Alpha one antitrypsin (α1AT), a serine proteinase inhibitor primarily produced by the liver, protects pulmonary tissue from neutrophil elastase digestion. Mutations of the SERPINA1 gene results in a misfolded α1AT protein which aggregates inside hepatocytes causing cellular damage. Therefore, inhibition of mutant α1AT production is one practical strategy to alleviate liver damage. Here we show that proteasome inhibitors can selectively downregulate α1AT expression in human hepatocytes by suppressing the translation of α1AT. Translational suppression of α1AT is mediated by phosphorylation of eukaryotic translation initiation factor 2α and increased association of RNA binding proteins, especially stress granule protein Ras GAP SH3 binding protein (G3BP1), with α1AT mRNA. Treatment of human-induced pluripotent stem cell-derived hepatocytes with a proteasome inhibitor also results in translational inhibition of mutant α1AT in a similar manner. Together we revealed a previously undocumented role of proteasome inhibitors in the regulation of α1AT translation.
... Despite bortezomib's clinical efficacy in treating hematologic malignancies, it has had limited success in clinical trials for solid tumors. This may arise from poor bortezomib tissue penetration (at the doses used in MM) and rapid clearance from the blood (Adams, 2002). Due to an increased risk for PN, the dosage cannot be increased to overcome poor tissue penetration. ...
The ubiquitin proteasome system (UPS) degrades individual proteins in a highly regulated fashion and is responsible for the degradation of misfolded, damaged, or unneeded cellular proteins. During the past 20 years, investigators have established a critical role for the UPS in essentially every cellular process, including cell cycle progression, transcriptional regulation, genome integrity, apoptosis, immune responses, and neuronal plasticity. At the center of the UPS is the proteasome, a large and complex molecular machine containing a multicatalytic protease complex. When the efficiency of this proteostasis system is perturbed, misfolded and damaged protein aggregates can accumulate to toxic levels and cause neuronal dysfunction, which may underlie many neurodegenerative diseases. In addition, many cancers rely on robust proteasome activity for degrading tumor suppressors and cell cycle checkpoint inhibitors necessary for rapid cell division. Thus, proteasome inhibitors have proven clinically useful to treat some types of cancer, especially multiple myeloma. Numerous cellular processes rely on finely tuned proteasome function, making it a crucial target for future therapeutic intervention in many diseases, including neurodegenerative diseases, cystic fibrosis, atherosclerosis, autoimmune diseases, diabetes, and cancer. In this review, we discuss the structure and function of the proteasome, the mechanisms of action of different proteasome inhibitors, various techniques to evaluate proteasome function in vitro and in vivo, proteasome inhibitors in preclinical and clinical development, and the feasibility for pharmacological activation of the proteasome to potentially treat neurodegenerative disease.
... Cependant le mécanisme est similaire a celui des protéases serine. Une caractéristique du protéasome provient du fait qu'il utilise la thréonine comme résidu catalytique [Adams, 2002]. ...
Lors des pathologies articulaires, les chondrocytes sont soumis à différents processus qui concourent à la dégradation du cartilage articulaire soit en entraînant la mort par apoptose (nonrenouvellement des constituants de la matrice) soit par l?activation de protéases détruisant les constituants matriciels. Le potentiel chondroprotecteur des protéines de stress (Hsp70 / Hsp27) lors des pathologies dégénératives ayant été démontré, nous avons souhaité évaluer l'intérêt thérapeutique de l'induction de ces protéines par un inhibiteur réversible du protéasome (MG132) dans un modèle expérimental : modèle par section du ligament croisé antérieur (SLCA). Au cours de cette étude, nous avons du évaluer un nouveau système de vectorisation afin de surexprimer les protéines de stress dans le cartilage articulaire. Cette technique (électroporation biphasique) repose sur la dissociation des impulsions perméabilisantes et électrophorétiques. Nous avons donc développé des plasmides nous permettant de surexprimer ces protéines, fusionnées à une protéine traceur, facilitant ainsi leur détection dans le tissu ciblé. Puis, nous avons évalué l'innocuité et l'efficacité des impulsions sur des tissus sains et pathologiques (dégénératifs et inflammatoires). Nous avons constaté que cette technique, en plus de limiter les altérations du tissu articulaire ou les phénomènes de dissémination, offrait des propriétés d'adressage tissulaire de part la multiplicité des combinaisons d'impulsions (nombre, fréquence et intensité). Enfin, les effets de l'induction (MG132) des Hsps sur un modèle physiopathologique (SLCA) ont été évalués et nous avons pu montrer une diminution de la sévérité des atteintes articulaires, tant au niveau du cartilage que de la membrane synoviale. Cette molécule non seulement permet à la fois de renforcer la résistance des chondrocytes aux atteintes mais également de limiter l'amplitude de la réponse inflammatoire qui participe à la dégradation.
... After the success of proteasomal inhibitors in the treatment of multiple myeloma, components of the UPS are being researched as drug targets against other malignancies as well as metabolic and neurodegenerative diseases. As a result, novel UPS enzymes and their adaptors have been identified as conceivable targets for the development of more selective and specific therapeutics (Adams, 2002;Orlowski et al., 2002;Nalepa and Harper, 2003;Issaeva et al., 2004;Hoeller et al., 2006;Richardson et al., 2006;Deshaies, 2014;Huang and Dixit, 2016). ...
Regulatory functions of the ubiquitin-proteasome system (UPS) are exercised mainly by the ubiquitin ligases and deubiquitinating enzymes. Degradation of apoptotic proteins by UPS is central to the maintenance of cell health, and deregulation of this process is associated with several diseases including tumors, neurodegenerative disorders, diabetes, and inflammation. Therefore, it is the view that interrogating protein turnover in cells can offer a strategy for delineating disease-causing mechanistic perturbations and facilitate identification of drug targets. In this review, we are summarizing an overview to elucidate the updated knowledge on the molecular interplay between the apoptosis and UPS pathways. We have condensed around 100 enzymes of UPS machinery from the literature that ubiquitinates or deubiquitinates the apoptotic proteins and regulates the cell fate. We have also provided a detailed insight into how the UPS proteins are able to fine-tune the intrinsic, extrinsic, and p53-mediated apoptotic pathways to regulate cell survival or cell death. This review provides a comprehensive overview of the potential of UPS players as a drug target for cancer and other human disorders.
... Targeting specific pathways prominently activated in EBV-associated DLBCL EBV-positive DLBCL of the elderly has an activated B-cell (ABC) DLBCL profile and is characterized by increased activation of the NF-kB pathway. Bortezomib inhibits the 26S proteasome, important in the degradation of the NF-kB inhibitor IkB [42] . In EBV transformed B-cells and mouse models, bortezomib has induced apoptosis [43] . ...
Epstein–Barr virus (EBV)‐positive diffuse large B‐cell lymphoma (DLBCL) of the elderly is a provisional entity included in the 2008 WHO classification of lymphoid neoplasms. It is a disease typically seen in the elderly and thought to be associated with chronic EBV infection and severe immunosuppression with a component of immunosenescence. Recent research, however, has suggested that EBV‐positive DLBCL can be seen in younger, immunocompetent patients.
The diagnosis of EBV‐positive DLBCL of the elderly is made through a careful pathological evaluation. The differential diagnosis includes infectious mononucleosis (specifically in younger patients), lymphomatoid granulomatosis, Hodgkin lymphoma, and gray zone lymphoma, among others. Detection of EBV‐encoded RNA (EBER) is considered standard for diagnosis; however, a clear cutoff for positivity has not been defined.
The International Prognostic Index (IPI), and the Oyama score can be used for risk‐stratification. The Oyama score includes age >70 years and presence of B symptoms. The expression of CD30 is emerging as a potential adverse, and targetable, prognostic factor.
Patients with EBV‐positive DLBCL should be staged and managed following similar guidelines than patients with EBV‐negative DLBCL. It has been suggested, however, that EBV‐positive patients have a worse prognosis than EBV‐negative counterparts in the era of chemoimmunotherapy. There is an opportunity to study and develop targeted therapy in the management of patients with EBV‐positive DLBCL. Am. J. Hematol. 91:530–537, 2016. © 2016 Wiley Periodicals, Inc.
... Therefore, the sites of the 26S proteasome, both 19S and 20S subunits, have been drug targets for various disease indications including cancer [4] . Bortezomib, a drug administered intravenously or subcutaneously , was the first proteasome inhibitor targeting enzymatic sites of the β chain and validated the proteasome as an anti-cancer target [5, 6]. It was approved for the treatment of mantle cell lymphoma and MM. ...
Background:
The proteasome is a validated anti-cancer target and various small-molecule inhibitors are currently in clinical development or on the market. However, adverse events and resistance associated with those proteasome inhibitors indicate the need for a new generation of drugs. Therefore, we focused on developing an oral proteasome inhibitor with improved efficacy and safety profiles.
Method:
The in vitro inhibition of the 20S proteasome catalytic activities was determined in human multiple myeloma (MM) cellular lysates with fluorogenic peptide substrates specific for each catalytic subunit. Cell cytotoxicity was assessed with the ATP bioluminescence assay using human cell samples from tumor cell lines, MM patients or normal healthy donors. In mice bearing human MM xenografts, a single dose of LC53-0110 was administered orally, and concentration-time profiles of LC53-0110 and the 20S proteasome catalytic activities in plasma, blood, and tumor were determined. The efficacy of repeat-dose compound with regard to tumor growth inhibition in vivo was also evaluated in the same MM xenograft models.
Results:
LC53-0110 is far more specific for the chymotrypsin-like proteolytic (β5) site of the 20S proteasome as compared to bortezomib, carfilzomib, or ixazomib. LC53-0110 treatment showed accumulation of ubiquitinated proteins, inhibited cell viability with a low nM range potency in various tumor cell lines, and showed potent activity on CD138(+) cells isolated from MM patients who are resistant/refractory to current FDA-approved drug treatment. When a single dose was administered orally to tumor-bearing mice, LC53-0110 showed both greater maximum and sustained tumor proteasome inhibition as compared with ixazomib in MM xenograft models. The robust pharmacodynamic responses in tumor correlated with tumor growth regression. In addition, LC53-0151, an analog of LC53-0110, in combination with pomalidomide, a third-generation immunomodulatory drug, showed synergistic inhibition of tumor growth both in vitro and in the xenograft mouse model.
Conclusions:
In view of the in vitro, in vivo, and ex vivo profiles, further investigation of additional LC compounds in preclinical studies is warranted for the nomination of a clinical development candidate.
... [16][17][18][19][20] Moreover, high-proteasome activity has been reported in different malignancies. 14 Because cancer cells are more sensitive than normal cells to the inhibition of proteasome activity, 21,22 targeting the proteasome pathway has emerged as an ideal approach in anticancer therapy. ...
Proteasome inhibitors are emerging as a new class of chemopreventive agents and have gained huge importance as potential pharmacological tools in breast cancer treatment. Improved understanding of the role played by proteases and their specific inhibitors in humans offers novel and challenging opportunities for preventive and therapeutic intervention. In this study, we demonstrated that the Bowman–Birk protease inhibitor from Vigna unguiculata seeds, named black-eyed pea trypsin/chymotrypsin Inhibitor (BTCI), potently suppresses human breast adenocarcinoma cell viability by inhibiting the activity of proteasome 20S. BTCI induced a negative growth effect against a panel of breast cancer cells, with a concomitant cytostatic effect at the G2/M phase of the cell cycle and an increase in apoptosis, as observed by an augmented number of cells at the sub-G1 phase and annexin V-fluorescin isothiocyanate (FITC)/propidium iodide (PI) staining. In contrast, BTCI exhibited no cytotoxic effect on normal mammary epithelial cells. Moreover, the increased levels of intracellular reactive oxygen species (ROS) and changes in the mitochondrial membrane potential in cells treated with BTCI indicated mitochondrial damage as a crucial cellular event responsible for the apoptotic process. The higher activity of caspase in tumoral cells treated with BTCI in comparison with untreated cells suggests that BTCI induces apoptosis in a caspase-dependent manner. BTCI affected NF-kB target gene expression in both non invasive and invasive breast cancer cell lines, with the effect highly pronounced in the invasive cells. An increased expression of interleukin-8 (IL-8) in both cell lines was also observed. Taken together, these results suggest that BTCI promotes apoptosis through ROS-induced mitochondrial damage following proteasome inhibition. These findings highlight the pharmacological potential and benefit of BTCI in breast cancer treatment.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Background
Bortezomib (BZM), alone or in combination with other chemotherapies, has displayed strong anticancer effects in several cancers. The efficacy of the combination of BZM and mitoxantrone (MTX) in treating prostate cancer remains unknown.
Methods
Anticancer effects of combination of BZM and MTX were determined by apoptosis and proliferation assay in vivo and in vitro. Expression of β-Catenin and its target genes were characterized by western blot and Real-time PCR.
Results
BZM significantly enhanced MTX-induced antiproliferation in vivo and in vitro. Mice administered a combination of BZM and MTX displayed attenuated tumor growth and prolonged survival. BZM significantly attenuated MTX-induced apoptosis. Moreover, the combination of BZM and MTX contributed to inhibition of the Wnt/β-Catenin signaling pathway compared to monotherapy.
Conclusions
This study demonstrates that BZM enhances MTX-induced anti-tumor effects by inhibiting the Wnt/β-Catenin signaling pathway in prostate cancer cells.
Background Bortezomib (BZM), alone or in combination with other chemotherapies, has displayed strong anticancer effects in several cancers. The efficacy of the combination of BZM and mitoxantrone (MTX) in treating prostate cancer remains unknown.
Methods Anticancer effects of combination of BZM and MTX were determined by apoptosis and proliferation assay in vivo and in vitro. Expression of β-catenin and its target genes were characterized by western blot and Real-time PCR.
Results BZM significantly enhanced MTX-induced antiproliferation in vivo and in vitro. Mice administered a combination of BZM and MTX displayed attenuated tumor growth and prolonged survival. BZM significantly attenuated MTX-induced apoptosis. Moreover, the combination of BZM and MTX contributed to inhibition of the Wnt/β-catenin signaling pathway compared to monotherapy.
Conclusions This study demonstrates that BZM enhances MTX-induced anti-tumor effects by inhibiting the Wnt/β-catenin signaling pathway in prostate cancer cells.
Proteasome inhibitors have become an important drug class in the treatment of multiple myeloma. In addition to its role in myeloma cells, the proteasome plays a critical role in the myocardium, particularly in the context of cardiac stress. The growing awareness of the cardiovascular toxicity of proteasome inhibitors is emerging following the phase 3 trials and the transition into real-world practice. This article reviews the background to this problem and the incidence of the problem in phase 3 trials and subsequent phase 2 trials in new patient cohorts and discusses the strategy to detect and manage this emerging problem.
Inhibition of deubiquitinase (DUB) activity is a promising strategy for cancer therapy. VLX1570 is an inhibitor of proteasome DUB activity currently in clinical trials for relapsed multiple myeloma. Here we show that VLX1570 binds to and inhibits the activity of ubiquitin-specific protease-14 (USP14) in vitro, with comparatively weaker inhibitory activity towards UCHL5 (ubiquitin-C-terminal hydrolase-5). Exposure of multiple myeloma cells to VLX1570 resulted in thermostabilization of USP14 at therapeutically relevant concentrations. Transient knockdown of USP14 or UCHL5 expression by electroporation of siRNA reduced the viability of multiple myeloma cells. Treatment of multiple myeloma cells with VLX1570 induced the accumulation of proteasome-bound high molecular weight polyubiquitin conjugates and an apoptotic response. Sensitivity to VLX1570 was moderately affected by altered drug uptake, but was unaffected by overexpression of BCL2-family proteins or inhibitors of caspase activity. Finally, treatment with VLX1570 was found to lead to extended survival in xenograft models of multiple myeloma. Our findings demonstrate promising antiproliferative activity of VLX1570 in multiple myeloma, primarily associated with inhibition of USP14 activity.
Based on the central role of the ubiquitin-proteasome system (UPS) in the degradation of cellular proteins, proteasome inhibition has been considered an attractive approach for anticancer therapy. Deubiquitinases (DUBs) remove ubiquitin conjugates from diverse substrates; therefore, they are essential regulators of the UPS. DUB inhibitors, especially the inhibitors of proteasomal DUBs are becoming a research hotspot in targeted cancer therapy. Previous studies have shown that metal complexes, such as copper and zinc complexes, can induce cancer cell apoptosis through inhibiting UPS function. Moreover, we have found that copper pyrithione inhibits both 19S proteasome-associated DUBs and 20S proteasome activity with a mechanism distinct from that of the classical 20S proteasome inhibitor bortezomib. In the present study, we reveal that (i) nickel pyrithione complex (NiPT) potently inhibits the UPS via targeting the 19S proteasome-associated DUBs (UCHL5 and USP14), without effecting on the 20S proteasome; (ii) NiPT selectively induces proteasome inhibition and apoptosis in cultured tumor cells and cancer cells from acute myeloid leukemia human patients; and (iii) NiPT inhibits proteasome function and tumor growth in nude mice. This study, for the first time, uncovers a nickel complex as an effective inhibitor of the 19S proteasomal DUBs and suggests a potentially new strategy for cancer treatment.Oncogene advance online publication, 18 April 2016; doi:10.1038/onc.2016.114.
Dynamic modulation of protein levels is tightly controlled in response to physiological cues. In mammalian cells, much of the protein degradation is carried out by the ubiquitin-proteasome system (UPS). Similar to kinases, components of the ubiquitin system are often dysregulated, leading to a variety of diseases, including cancer and neurodegeneration, making them attractive drug targets. However, so far there are only a handful of drugs targeting the ubiquitin system that have been approved by the FDA. Here, we review possible therapeutic intervention nodes in the ubiquitin system, analyze the challenges, and highlight the most promising strategies to target the UPS.Cell Research advance online publication 22 March 2016; doi:10.1038/cr.2016.31.
The transcription factors of the Rel/NF-κB family are key regulators of immune and inflammatory responses and contribute to
lymphocyte proliferation, survival, and oncogenesis. The absolute correlation between the antiapoptotic and oncogenic activities
of the Rel/NF-κB oncoprotein v-Rel emphasizes the importance of characterizing the death antagonists under NF-κB control.
Our recent finding that the prosurvival Bcl-2 homolog Bfl-1 (also called A1) is a direct transcriptional target of NF-κB raised
the issue of whether NF-κB is a specific or global regulator of death antagonists in the Bcl-2 family. Here, we demonstrate
that NF-κB differentially regulates the expression of particular Bcl-2-related death inhibitors and that it directly activates
the expression of Bcl-xL. While Bcl-xL was significantly upregulated by c-Rel and RelA, Bcl-2 was not. Importantly, stimuli that activate endogenous NF-κB factors
also upregulated bcl-x gene expression and this effect was antagonized by an inhibitor of NF-κB activity. The expression of bcl-x suppressed apoptosis in the presence or absence of NF-κB activity. Functional analysis of thebcl-x promoter demonstrated that it is directly controlled by c-Rel. These results establish that NF-κB directly regulates the
expression of distinct prosurvival factors in the Bcl-2 family, such as Bcl-xL and Bfl-1/A1. These findings raise the possibility that some of these factors may contribute to oncogenesis associated with
aberrant Rel/NF-κB activity.
Proteasomes are eukaryotic ring-shaped or cylindrical particles with multicatalytic protease activities. To clarify the involvement of proteasomes in tumorigenesis of human blood cells, we compared their expression in human hematopoietic malignant tumor cells with that in normal peripheral blood mononuclear cells. Immunohistochemical staining showed considerably increased concentrations of proteasomes in leukemic cells from the bone marrow of patients with various types of leukemia and the predominant localization of these proteasomes in the nuclei. Moreover, enzyme immunoassay and Northern blot analysis indicated that the concentrations of proteasomes and their mRNA levels were consistently much higher in a variety of malignant human hematopoietic cell lines than in resting peripheral lymphocytes and monocytes from healthy adults. Proteasome expression was also greatly increased in normal blood mononuclear cells during blastogenic transformation induced by phytohemagglutinin; their expression increased in parallel with induction of DNA synthesis and returned to the basal level with progress of the cell cycle. Thus, abnormally high expression of proteasomes may play an important role in transformation and proliferation of blood cells and in specific functions of hematopoietic tumor cells.
Lactacystin is a Streptomyces metabolite that inhibits cell cycle progression and induces neurite outgrowth in a murine neuroblastoma
cell line. Tritium-labeled lactacystin was used to identify the 20S proteasome as its specific cellular target. Three distinct
peptidase activities of this enzyme complex (trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolyzing activities)
were inhibited by lactacystin, the first two irreversibly and all at different rates. None of five other proteases were inhibited,
and the ability of lactacystin analogs to inhibit cell cycle progression and induce neurite outgrowth correlated with their
ability to inhibit the proteasome. Lactacystin appears to modify covalently the highly conserved amino-terminal threonine
of the mammalian proteasome subunit X (also called MB1), a close homolog of the LMP7 proteasome subunit encoded by the major
histocompatibility complex. This threonine residue may therefore have a catalytic role, and subunit X/MB1 may be a core component
of an amino-terminal-threonine protease activity of the proteasome.
Reagents that inhibit the ubiquitin-proteasome proteolytic pathway in cells have not been available. Peptide aldehydes that inhibit major peptidase activities of the 20S and 26S proteasomes are shown to reduce the degradation of protein and ubiquitinated protein substrates by 26S particles. Unlike inhibitors of lysosomal proteolysis, these compounds inhibit the degradation of not only abnormal and short-lived polypeptides but also long-lived proteins in intact cells. We used these agents to test the importance of the proteasome in antigen presentation. When ovalbumin is introduced into the cytosol of lymphoblasts, these inhibitors block the presentation on MHC class I molecules of an ovalbumin-derived peptide by preventing its proteolytic generation. By preventing peptide production from cell proteins, these inhibitors block the assembly of class I molecules. Therefore, the proteasome catalyzes the degradation of the vast majority of cell proteins and generates most peptides presented on MHC class I molecules.
Lactacystin is a Streptomyces metabolite that inhibits cell cycle progression and induces differentiation in a murine neuroblastoma cell line. The cellular target of lactacystin is the 20 S proteasome, also known as the multicatalytic proteinase complex, an essential component of the ubiquitin-proteasome pathway for intracellular protein degradation. In aqueous solution at pH 8, lactacystin undergoes spontaneous hydrolysis to yield N-acetyl-L-cysteine and the inactive lactacystin analog, clasto-lactacystin dihydroxy acid. We have studied the mechanism of lactacystin hydrolysis under these conditions and found that it proceeds exclusively through the intermediacy of the active lactacystin analog, clasto-lactacystin beta-lactone. Conditions that stabilize lactacystin (and thus prevent the transient accumulation of the intermediate beta-lactone) negate the ability of lactacystin to inactivate the proteasome. Together these findings suggest that lactacystin acts as a precursor for clasto-lactacystin beta-lactone and that the latter is the sole species that interacts with the proteasome.
Activation of proteolytic enzymes, including cysteine proteases of the ced-3/ICE family, is a characteristic feature of the apoptotic program. In contrast, the role of the proteasome as the major nonlysosomal machinery to degrade or process proteins by ATP/ubiquitin-dependent proteolysis in this process is less clear. In human leukemic HL60 cells, inhibition of proteasome-mediated proteolysis by specific proteasomal inhibitors leads to the rapid induction of apoptosis as judged by morphological changes as well as by nuclear condensation and DNA fragmentation. HL60 apoptosis is due to activation of CPP32, a member of the ced-3/ICE family of cysteine proteases, and appears to occur independently from ICE activity. HL60 apoptosis is accompanied by an increase in the concentration of the cyclin-dependent kinase inhibitor p27Kip1. Labeling of the cells by the TUNEL technique demonstrates that HL60 cells undergoing apoptosis are primarily in the G1 phase of the cell cycle. Proteasomal activity therefore appears to be required in proliferating, but not in quiescent, HL60 cells for cell survival as well as normal progression through the cell cycle.
The proteasome is a multicatalytic protease complex that plays a key role in diverse cellular functions. The peptide vinyl sulfone, carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone (Z-L3VS) covalently inhibits the trypsin-like, chymotrypsin-like and, unlike lactacystin, also the peptidylglutamyl peptidase activity in isolated proteasomes, and blocks their function in living cells. Although described as a class of mechanism-based inhibitors for cysteine proteases, the peptide vinyl sulfone Z-L3VS and a 125I-labeled nitrophenol derivative (125I-NIP-L3VS) covalently modify the active site threonine of the catalytic beta subunits of the proteasome. Modification of Thermoplasma proteasomes demonstrates the requirement for a hydroxyl amino acid (threonine, serine) as nucleophile at the beta subunit's NH2 terminus. 125I-NIP-L3VS covalently modifies the HslV subunit of the Escherichia coli protease complex HslV/HslU, a reaction that requires ATP, and supports a catalytic mechanism shared with that of the eukaryotic proteasome.
The 436-amino acid protein enolase 1 from yeast was degraded in vitro by purified wild-type and mutant yeast 20S proteasome particles. Analysis of the cleavage products at different times revealed a processive degradation mechanism and a length distribution of fragments ranging from 3 to 25 amino acids with an average length of 7 to 8 amino acids. Surprisingly, the average fragment length was very similar between wild-type and mutant 20S proteasomes with reduced numbers of active sites. This implies that the fragment length is not influenced by the distance between the active sites, as previously postulated. A detailed analysis of the cleavages also allowed the identification of certain amino acid characteristics in positions flanking the cleavage site that guide the selection of the P1 residues by the three active beta subunits. Because yeast and mammalian proteasomes are highly homologous, similar cleavage motifs might be used by mammalian proteasomes. Therefore, our data provide a basis for predicting proteasomal degradation products from which peptides are sampled by major histocompatibility complex class I molecules for presentation to cytotoxic T cells.
The transcription factor NF-kappaB activates a number of genes whose protein products are proinflammatory. In quiescent cells, NF-kappaB exists in a latent form and is activated via a signal-dependent proteolytic mechanism in which the inhibitory protein IkappaB is degraded by the ubiquitin-proteasome pathway. Consequently, inhibition of the proteasome suppresses activation of NF-kappaB. This suppression should therefore decrease transcription of many genes encoding proinflammatory proteins and should ultimately have an anti-inflammatory effect. To this end, a series of peptide boronic acid inhibitors of the proteasome, exemplified herein by PS-341, were developed. The proteasome is the large multimeric protease that catalyzes the final proteolytic step of the ubiquitin-proteasome pathway. PS-341, a potent, competitive inhibitor of the proteasome, readily entered cells and inhibited the activation of NF-kappaB and the subsequent transcription of genes that are regulated by NF-kappaB. Significantly, PS-341 displayed similar effects in vivo. Oral administration of PS-341 had anti-inflammatory effects in a model of Streptococcal cell wall-induced polyarthritis and liver inflammation in rats. The attenuation of inflammation in this model was associated with an inhibition of IkappaBalpha degradation and NF-kappaB-dependent gene expression. These experiments clearly demonstrate that the ubiquitin-proteasome pathway and NF-kappaB play important roles in regulating chronic inflammation and that, as predicted, proteasome inhibition has an anti-inflammatory effect.
Bcl-2-family proteins are key regulators of the apoptotic response. Here, we demonstrate that the pro-survival Bcl-2 homolog Bfl-1/A1 is a direct transcriptional target of NF-kappaB. We show that bfl-1 gene expression is dependent on NF-kappaB activity and that it can substitute for NF-kappaB to suppress TNFalpha-induced apoptosis. bfl-1 promoter analysis identified an NF-kappaB site responsible for its Rel/NF-kappaB-dependent induction. The expression of bfl-1 in immune tissues supports the protective role of NF-kappaB in the immune system. The activation of Bfl-1 may be the means by which NF-kappaB functions in oncogenesis and promotes cell resistance to anti-cancer therapy.
The ubiquitin-proteasome pathway plays a critical role in the regulated degradation of proteins involved in cell cycle control and tumor growth. Dysregulating the degradation of such proteins should have profound effects on tumor growth and cause cells to undergo apoptosis. To test this hypothesis, we developed a novel series of proteasome inhibitors, exemplified by PS-341, which we describe here. As determined by the National Cancer Institute in vitro screen, PS-341 has substantial cytotoxicity against a broad range of human tumor cells, including prostate cancer cell lines. The PC-3 prostate cell line was, therefore, chosen to further examine the antitumor activity of PS-341. In vitro, PS-341 elicits proteasome inhibition, leading to an increase in the intracellular levels of specific proteins, including the cyclin-dependent kinase inhibitor, p21. Moreover, exposure of such cells to PS-341 caused them to accumulate in the G2-M phase of the cell cycle and subsequently undergo apoptosis, as indicated by nuclear condensation and poly(ADP-ribose) polymerase cleavage. Following weekly i.v. treatment of PS-341 to mice bearing the PC-3 tumor, a significant decrease (60%) in tumor burden was observed in vivo. Direct injection of PS-341 into the tumor also caused a substantial (70%) decrease in tumor volume with 40% of the drug-treated mice having no detectable tumors at the end of the study. Studies also revealed that i.v. administration of PS-341 resulted in a rapid and widespread distribution of PS-341, with highest levels identified in the liver and gastrointestinal tract and lowest levels in the skin and muscle. Modest levels were found in the prostate, whereas there was no apparent penetration of the central nervous system. An assay to follow the biological activity of the PS-341 was established and used to determine temporal drug activity as well as its ability to penetrate tissues. As such, PS-341 was shown to penetrate PC-3 tumors and inhibit intracellular proteasome activity 1.0 h after i.v. dosing. These data illustrate that PS-341 not only reaches its biological target but has a direct effect on its biochemical target, the proteasome. Importantly, the data show that inhibition of this target site by PS-341 results in reduced tumor growth in murine tumor models. Together, the results highlight that the proteasome is a novel biochemical target and that inhibitors such as PS-341 represent a unique class of antitumor agents. PS-341 is currently under clinical evaluation for advanced cancers.
The proteasome regulates cellular processes as diverse as cell cycle progression and NF-κB activation. In this study, we show
that the potent antitumor natural product epoxomicin specifically targets the proteasome. Utilizing biotinylated-epoxomicin
as a molecular probe, we demonstrate that epoxomicin covalently binds to the LMP7, X, MECL1, and Z catalytic subunits of the
proteasome. Enzymatic analyses with purified bovine erythrocyte proteasome reveal that epoxomicin potently inhibits primarily
the chymotrypsin-like activity. The trypsin-like and peptidyl-glutamyl peptide hydrolyzing catalytic activities also are inhibited
at 100- and 1,000-fold slower rates, respectively. In contrast to peptide aldehyde proteasome inhibitors, epoxomicin does
not inhibit nonproteasomal proteases such trypsin, chymotrypsin, papain, calpain, and cathepsin B at concentrations of up
to 50 μM. In addition, epoxomicin is a more potent inhibitor of the chymotrypsin-like activity than lactacystin and the peptide
vinyl sulfone NLVS. Epoxomicin also effectively inhibits NF-κB activation in vitro and potently blocks in vivo inflammation in the murine ear edema assay. These results thus define epoxomicin as a novel proteasome inhibitor that likely
will prove useful in exploring the role of the proteasome in various in vivo and in vitro systems.
The response of a cell to its external environment requires rapid and significant alteration of protein amount, localization and/or function. This regulation involves a complex combination of processes that control synthesis, localization and degradation. All of these processes must be properly regulated and are often interrelated. Intracellular proteolysis is largely accomplished by the ubiquitin-dependent system and has been shown to be required for growth control, cell cycle regulation, receptor function, development and the stress response. Substrates subject to regulated degradation by this system include cyclins and cyclin-dependent kinase inhibitors, tumor suppressors, transcription factors and cell surface receptors. In addition, proteins that are damaged by oxidation or that are improperly folded or localized are substrates whose degradation by this system often leads to antigen presentation on the surface of the cell in the context of Class I major histocompatibility complex molecules. A very large body of work in the last fifteen years has shown that degradation by this system requires the covalent attachment of a small protein called ubiquitin and that this modification serves to direct target proteins for degradation by a 26S proteolytic particle, the proteasome. Thus, the attachment of the ubiquitin domain is of vital importance in regulating normal growth and differentiation, as well as in defending against cellular damage caused by xenobiotics, environmental insults, infection and mutation. This review focuses on the role of ubiquitination in the cellular signaling pathways that deal with these external influences.
The pleiotropic transcription factor NF-kappaB controls cellular apoptotic and growth processes and increasing evidence suggests a role in tumorigenesis. We describe here that constitutively activated NF-kappaB complexes are found in the vast majority (39 out of 42 samples) of childhood acute lymphoblastic leukemia (ALL) without any subtype restriction. Electrophoretic shift analysis further demonstrates that these complexes are composed of p50-p50 and p65-p50 dimers. Proteasome inhibition in primary ALL cultures results in a hyperphosphorylated form of IkappaBalpha, indicating that activation of upstream kinases, which trigger IkappaBalpha degradation, has led to nuclear translocation of NF-kappaB. Careful inhibition of cellular proteolytic activities is of importance when analyzing extracts from primary ALL cells. Degradation of p65 and other proteins in ALL samples could be specifically suppressed by alpha-1 antitrypsin. Constitutive NF-kappaB activation is thus a common characteristic of childhood ALL and strongly suggests a critical role of this factor for leukemia cell survival.
The proteasome is a multiprotein complex that is involved in the intracellular protein degradation in eukaryotes. Here, we show that human malignant glioma cells are susceptible to apoptotic cell death induced by the proteasome inhibitors, MG132 and lactacystin. The execution of the apoptotic death program involves the processing of caspases 2, 3, 7, 8, and 9. Apoptosis is inhibited by ectopic expression of X-linked inhibitor of apoptosis (XIAP) and by coexposure to the broad-spectrum caspase inhibitor, benzoyl-VAD-fluoromethyl ketone (zVAD-fmk), but not by the preferential caspase 8 inhibitor, crm-A. It is interesting that specific morphological alterations induced by proteasome inhibition, such as dilated rough endoplasmic reticulum and the formation of cytoplasmic vacuoles and dense mitochondrial deposits, are unaffected by zVAD-fmk. Apoptosis is also inhibited by ectopic expression of Bcl-2 or by an inhibitor of protein synthesis, cycloheximide. Further, cytochrome c release and disruption of mitochondrial membrane potential are prominent features of apoptosis triggered by proteasome inhibition. Bcl-2 is a stronger inhibitor of cytochrome c release than zVAD-fmk. XIAP and crm-A fail to modulate cytochrome c release. These data place cytochrome c release downstream of Bcl-2 activity but upstream of XIAP- and crm-A-sensitive caspases. The partial inhibition of cytochrome c release by zVAD-fmk indicates a positive feedback loop that may involve cytochrome c release and zVAD-fmk-sensitive caspases. Finally, death ligand/receptor interactions, including the CD95/CD95 ligand system, do not mediate apoptosis induced by proteasome inhibition in human malignant glioma cells.
We have examined the effects of inhibition of the 26S proteasome in a murine mammary cell line, KIM-2 cells using the peptide aldehyde inhibitor MG132. These studies have demonstrated a clear requirement for proteasome function in cell viability. Induction of apoptosis was observed following MG132 treatment in KIM-2 cells and this death was shown to be dependent on the cell actively traversing the cell cycle. KIM-2 cells were generated using a temperature sensitive T-antigen (Tag) and studies at the permissive temperature (33 degrees C) have shown that a Tag binding protein was essential for this apoptotic response. Studies in two additional cell lines, HC11, which is a mammary epithelial cell line carrying mutant p53 alleles and p53 null ES cells suggest that p53 is actively required for the apoptosis induced as a consequence of proteasome inhibition. These results suggest a pivotal role for the 26S proteasome degradation pathway in progression through the cell cycle in proliferating cells.
Degradation of several intracellular proteins involved in cell cycle control and tumour growth is regulated by the ubiquitin-dependent multicatalytic protease complex (proteasome). We report that proteasome inhibitor Z-Ile-Glu(OtBu)-Ala-Leucinal (PSI) was cytotoxic on most human myeloid leukaemia cell lines at IC50 doses ranging from 5 to 25 nmol/l. Additionally, PSI pre-treatment enhanced cytotoxicity by taxol and cisplatinum. PSI was more active on leukaemic than on normal CD34(+) bone marrow progenitors because the 50% growth inhibition of colony-forming unit granulocyte macrophage (CFU-GM) from cases of chronic myelogenous leukaemia (CML) and normal subjects was achieved by 15 nmol/l and 50 nmol/l PSI respectively. PSI killed cells by apoptosis as revealed by ultrastructural changes, nuclear DNA fragmentation, cleavage of poly (ADP-ribose) polymerase (PARP) and of beta-catenin, and was antagonized by ectopic expression of Bcl-2 but not by inactivating mutations of p53. This event was associated with a slight accumulation of Bcl-2, a decrease of Bax but no changes in Bcl-X(L) protein expression at any time point. In Ph(+) cell lines BCR-ABL protein was only down-regulated after 48 h of treatment with 10 nmol/l PSI. Altogether, these results indicate that PSI, alone or in association with other cytotoxic agents, has anti-tumour activity against myeloid malignancies and is more effective on leukaemic than on normal haematopoietic progenitor cells.
In this study, we investigated possible engagement of NF-kappaB and Ku autoantigen (Ku) activation in development of multidrug resistance (MDR) and circumvention of MDR by modulation of NF-kappaB and Ku. The NF-kappaB activity and NF-kappaB p65 subunit level were constitutively higher in MDR cells than in drug-sensitive parental cells. Interestingly, a faster running NF-kappaB DNA binding complex was identified as Ku, a DNA damage sensor and a key double strand break repair protein, and was positively correlated with the NF-kappaB activity in MDR cells and Ku- or both subunits of NF-kappaB-transfected cells. Also both NF-kappaB and Ku activities were activated or inhibited by treatment with etoposide (VP-16) or MG-132 (a proteasome inhibitor), respectively. Furthermore, PKA inhibitor suppressed markedly the constitutive and drug-induced activities of NF-kappaB and Ku in MDR cells and subsequently potentiated the cytotoxic activity of anticancer drugs. Our results proposed that the NF-kappaB and Ku activation could be one of multi-factorial MDR mechanism, and PKA inhibitor, likely via inhibition of NF-kappaB and Ku activities, could enhance the effectiveness of anticancer drugs against MDR cells with high activities of NF-kappaB and Ku.
Death-inducing ligands (DILs) such as tumor necrosis factor α (TNFα) or the cytotoxic drug doxorubicin have been shown to activate a nuclear factor κB (NFκB)-dependent program that may rescue cells from apoptosis induction. We demonstrate here that TRAIL (TNF-related apoptosis-inducing ligand), a recently identified DIL, also activates NFκB in lymphoid cell lines in a kinetic similar to TNFα. NFκB activity is independent from FADD, caspases, and apoptosis induction. To study the influence of NFκB activity on apoptosis mediated by TRAIL, CD95, TNFα, or doxorubicin, NFκB activation was inhibited using the proteasome inhibitor N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal or transient overexpression of mutant IκBα. Sensitivity for induction of apoptosis was markedly increased by these treatments in apoptosis sensitive cell lines. Moreover, both in cell lines and in primary leukemia cells that are resistant towards induction of apoptosis by DILs and doxorubicin, antagonization of NFκB activity partially restored apoptosis sensitivity. These data suggest that inhibition of NFκB activation may provide a molecular approach to increase apoptosis sensitivity in anticancer treatment.
Lactacystin was isolated from the culture broth of Streptomyces lactacystinaeus as an inducer of neurite outgrowth in Neuro 2a cells (a mouse neuroblastoma cell line). The structure of lactacystin, elucidated by spectroscopic analyses including NMR and X-ray crystallography, possesses a non-peptide skeleton consisting of two α-amino acids, N-acetylcysteine and a novel pyroglutamic acid derivative. Extensive studies on its mode of action revealed that lactacystin inhibits proteasome, a high molecular weight, multicatalytic protease complex responsible for most non-lysosomal intracellular protein degradation, by binding covalently to the active site N-terminal threonine residue in certain β-subunits of proteasome. Lactacystin and its cell-permeable β-lactone form, later designated omuralide by Prof. E. J. Corey, which are structurally different from the synthetic peptide aldehydes, are much more specific proteasome inhibitors. The demonstration of this lactacystin action gave decisive understanding of proteasome as a novel threonine protease. Since then, specific inhibitors have allowed researchers to simplify studies of proteasome functions, leading to many unexpected findings about the importance of the ubiquitin-proteasome pathway in various cellular processes, such as cell cycle, apoptosis, antigen presentation and the degradation of regulatory or membrane proteins. In this review, potential biomedical applications are also described.
The ubiquitin-proteasome pathway plays a critical role in the degradation of cellular proteins and cell cycle control. Dysregulating the degradation of such proteins should have profound effects on tumor growth and causes cells to undergo apoptosis. The aims of this study are to evaluate the ubiquitin-proteasome pathway in gastric cancer and the potential role of pharmacological inhibition of proteasome on induction of apoptosis in gastric cancer cells. Gastric cancer cell lines AGS (p53 wild-type) and MKN-28 (p53 mutant) were treated with proteasome inhibitor MG132. The results showed that MG132 inhibited cell proliferation in AGS and MKN-28 cells in a time- and dose-dependent manner. The inhibition of cell proliferation was caused by apoptosis which was also time- and dose-dependent. AGS cells were more responsive to MG132 than MKN-28 cells. Induction of apoptosis was preceded by the activation of caspase-3, as measured by a colorimetric caspase-3 cellular activity and Western blotting of the cleavage of caspase-3 and its substrate PARP. Activation of caspase-7 was also exhibited. In addition, z-VAD-fmk, a broad spectrum caspase inhibitor, reversed apoptosis induced by MG132 in AGS and MKN28 cells. Although z-DEVD-fmk, a specific caspase-3 inhibitor, suppressed MG132-induced apoptosis in MKN28 cells, it only partially rescued the apoptotic effect in AGS cells. Caspase-3 activation was the result of release of cytochrome c from mitochondria into the cytosol, as a consequence of upregulation of bax. There were overexpressions of all the proteasome-related proteins p53, p21waf1 and p27kip1 at 4 hr after proteasome inhibition which was identified by the accumulation of ubiquitin-tagged proteins. This was accompanied by accumulation of cells at G1 phase. Our present study suggests that inhibition of proteasome function in gastric cancer cells induces apoptosis and proteasomal inhibitors have potential use as novel anticancer drugs in gastric cancer.
The proteasome pathway is important for the turnover of many regulatory proteins. This pathway has recently become a target for antitumor agents and several research groups have demonstrated that inhibitors with specificities for the proteasome are potent apoptosis-inducing agents. Many mechanisms by which proteasome inhibitors exert their effects have been suggested, including inhibition of NF-κB activity and stabilization of the p53 tumor suppressor protein. We investigated the ability of inhibitors with specificities for the proteasome and for another protein degradation enzyme, calpain, to sensitize a murine B-cell lymphoma with constitutive NF-κB1 homodimer activity and high expression of Bcl-2 protein to radiation-induced apoptosis. Protease inhibitors tested were calpain inhibitor I, calpain inhibitor II, calpeptin, MG132, and Lactacystin. All five inhibitors induced apoptosis and sensitized cells to radiation despite the maintenance of Bcl-2 protein levels throughout the course of treatment. An electrophoretic migration shift assay for NF-κB1 activity provided evidence that reversal of NF-κB activity was not required for induction of cell death; however, p53 levels were elevated for all inhibitors tested. HL-60 cells, devoid of p53, could not be sensitized to radiation by MG132 treatment, suggesting that p53 was important for cell death induced by combined treatment with protease inhibitors and radiation. We concluded that protease inhibitors are capable of overcoming the protective effects of Bcl-2 to induce apoptosis and suggest that protease inhibitor treatment, when combined with ionizing radiation, leads to p53-mediated apoptosis. © 2001 Wiley-Liss, Inc.
The 26S proteasome is a non-lysosomal multicatalytic protease complex for degrading intracellular proteins by ATP/ubiquitin-dependent proteolysis. Tightly ordered proteasomal degradation of proteins critical for cell cycle control implies a role of the proteasome in maintaining cell proliferation and cell survival. In this study, we demonstrate that cell-permeable proteasome inhibitors, lactacystin, benzyloxycarbonyl(Z)-leucyl-leucyl-leucinal (ZLLLal; MG-132) and4-hydroxy-5-iodo-3-nitrophenylacetyl-leucyl-leucyl-leucine vinyl sulfone (NLVS), induce apoptosis abundantly in p53-defective leukemic cell lines CCRF-CEM, U937 and K562 as well as in myelogenic and lymphatic leukemic cells obtained from adult individuals with relapsed acute leukemias. Leukemic cell apoptosis induced by the proteasome inhibitors was dependent on activation of caspase-3 and related caspase family proteases, because caspase-3 inhibitor N-acetyl-l-aspartyl-l-glutamyl-l-valyl-l-aspartal (Ac-DEVD-cho) and, more effectively, the general caspase-inhibitor N-benzyloxycarbonyl-l-valyl-l-alanyl-l-aspartate fluoromethylketone (Z-VAD-fmk) were capable of blocking apoptosis induced by lactacystin, ZLLLal or NLVS. Induction of apoptosis by lactacystin or ZLLLal was accompanied by cell cycle arrest at G2/M phase and by accumulation and stabilization of cyclin-dependent kinase inhibitor p21WAF1/Cip and tumor suppressor protein p53. A role of p53 in mediating apoptosis or induction of p21WAF1/Cip1 was ruled out since CCRF-CEM and U937 cells express non-functional mutant p53, and K562 cells lack expression of p53. Viability and hematopoietic outgrowth of human CD34+ progenitor cells treated with lactacystin were slightly reduced, whereas treatment of CD34+ cells with ZLLLal or the cytostatic drugs doxorubicin and gemcitabine resulted in markedly reduced viability and hematopoietic outgrowth. These results demonstrate a basic role of the proteasome in maintaining survival of human leukemic cells, and may define cell-permeable proteasome inhibitors as potently anti-leukemic agents which exhibit a moderate hematopoietic toxicity in vitro.
Recent evidence indicates that the transcription factor NF-κB is a major effector of inducible antiapoptotic mechanisms. For example, it was shown that NF-κB activation suppresses the activation of caspase 8, the apical caspase in tumor necrosis factor (TNF) receptor family signaling cascades, through the transcriptional regulation of certain TRAF and IAP proteins. However, it was unknown whether NF-κB controls other key regulatory mechanisms in apoptosis. Here we show that NF-κB activation suppresses mitochondrial release of cytochrome
c
through the activation of the Bcl-2 family member A1/Bfl-1. The restoration of A1 in NF-κB null cells diminished TNF-induced apoptosis by reducing the release of proapoptotic cytochrome
c
from mitochondria. In addition, A1 potently inhibited etoposide-induced apoptosis by inhibiting the release of cytochrome
c
and by blocking caspase 3 activation. Our findings demonstrate that A1 is an important antiapoptotic gene controlled by NF-κB and establish that the prosurvival function of NF-κB can be manifested at multiple levels.
Death-inducing ligands (DILs) such as tumor necrosis factor alpha (TNFalpha) or the cytotoxic drug doxorubicin have been shown to activate a nuclear factor kappaB (NFkappaB)-dependent program that may rescue cells from apoptosis induction. We demonstrate here that TRAIL (TNF-related apoptosis-inducing ligand), a recently identified DIL, also activates NFkappaB in lymphoid cell lines in a kinetic similar to TNFalpha. NFkappaB activity is independent from FADD, caspases, and apoptosis induction. To study the influence of NFkappaB activity on apoptosis mediated by TRAIL, CD95, TNFalpha, or doxorubicin, NFkappaB activation was inhibited using the proteasome inhibitor N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal or transient overexpression of mutant IkappaBalpha. Sensitivity for induction of apoptosis was markedly increased by these treatments in apoptosis sensitive cell lines. Moreover, both in cell lines and in primary leukemia cells that are resistant towards induction of apoptosis by DILs and doxorubicin, antagonization of NFkappaB activity partially restored apoptosis sensitivity. These data suggest that inhibition of NFkappaB activation may provide a molecular approach to increase apoptosis sensitivity in anticancer treatment.
Tumor necrosis factor α (TNF-α) binding to the TNF receptor (TNFR) potentially initiates apoptosis and activates the transcription
factor nuclear factor kappa B (NF-κB), which suppresses apoptosis by an unknown mechanism. The activation of NF-κB was found
to block the activation of caspase-8. TRAF1 (TNFR-associated factor 1), TRAF2, and the inhibitor-of-apoptosis (IAP) proteins
c-IAP1 and c-IAP2 were identified as gene targets of NF-κB transcriptional activity. In cells in which NF-κB was inactive,
all of these proteins were required to fully suppress TNF-induced apoptosis, whereas c-IAP1 and c-IAP2 were sufficient to
suppress etoposide-induced apoptosis. Thus, NF-κB activates a group of gene products that function cooperatively at the earliest
checkpoint to suppress TNF-α–mediated apoptosis and that function more distally to suppress genotoxic agent–mediated apoptosis.
Potent and selective dipeptidyl boronic acid proteasome inhibitors are described. As compared to peptidyl aldehyde compounds, boronic acids in this series display dramatically enhanced potency. Compounds such as 15 are promising new therapeutics for treatment of cancer and inflammatory diseases.
The anticancer activity of the boronic acid dipeptide proteasome inhibitor PS-341 was examined in vitro and in vivo. PS-341 was a potent cytotoxic agent toward MCF-7 human breast carcinoma cells in culture, producing an IC90 of 0.05 microM on 24 h of exposure to the drug. In the EMT-6 tumor cell survival assay, PS-341 was equally cytotoxic administered p.o. or by i.p. injection up to a dose of 2 mg/kg. PS-341 was also toxic to the bone marrow colony-forming unit-granulocyte macrophage. PS-341 increased the tumor cell killing of radiation therapy, cyclophosphamide, and cisplatin in the EMT-6/Parent tumor, but was not able to overcome the in vivo resistance of the EMT-6/CTX and EMT-6/CDDP tumors. In the tumor growth delay assay, PS-341 administered p.o. had antitumor activity against the Lewis lung carcinoma, both primary and metastatic disease. In combination, regimens with 5-fluorouracil, cisplatin, Taxol and adriamycin, PS-341 seemed to produce primarily additive tumor growth delays against the s.c. tumor and was highly effective against disease metastatic to the lungs. The proteasome is an interesting new target for cancer therapy, and the proteasome inhibitor PS-341 warrants continued investigation in cancer therapy.
In eukaryotic cells, the vast majority of proteins in the cytosol and nucleus are degraded via the proteasome-ubiquitin pathway. The 26S proteasome is a huge protein degradation machine of 2.5 MDa, built of approximately 35 different subunits. It contains a proteolytic core complex, the 20S proteasome and one or two 19S regulatory complexes which associate with the termini of the barrel-shaped 20S core. The 19S regulatory complex serves to recognize ubiquitylated target proteins and is implicated to have a role in their unfolding and translocation into the interior of the 20S complex where they are degraded into oligopeptides. While much progress has been made in recent years in elucidating the structure, assembly and enzymatic mechanism of the 20S complex, our knowledge of the functional organization of the 19S regulator is rather limited. Most of its subunits have been identified, but specific functions can be assigned to only a few of them.
Proteolysis mediated by the ubiquitin-proteasome system has been implicated in the regulation of programmed cell death. Here we investigated the differential effects of proteasomal inhibitors on the viability of proliferating and quiescent primary endothelial cells in vitro and in vivo. Subconfluent, proliferating cells underwent carbobenzoxy-L-isoleucyl-gamma-t-butyl-L-glutamyl-L-alanyl-L-leucinal (PSI) -induced apoptosis at low concentrations (EC(50)=24 nM), whereas at least 340-fold higher concentrations of PSI were necessary to obtain the same effect in confluent, contact-inhibited cells. PSI-mediated cell death could be blocked by a caspase-3 inhibitor (Ac-DEVD-H), but not by a caspase-1 inhibitor (Ac-YVAD-H), suggesting that a caspase-3-like enzyme is activated during PSI-induced apoptosis. When applied to the embryonic chick chorioallantoic membrane, a rapidly expanding tissue, PSI induced massive apoptosis also in vivo. PSI treatment of the CAM led to the formation of areas devoid of blood flow due to the induction of apoptosis in endothelial and other cells and to the collapse of capillaries and first order vessels. Our results demonstrate that proteasomal inhibitors such as PSI may prove effective as novel anti-angiogenic and anti-neoplastic substances.
In search for angiogenesis inhibitors, we tested protease and proteasome inhibitors for the induction of G1 arrest and selective inhibition of growth of human umbilical vein endothelial cells (HUVECs). Serine protease-, cysteine protease-, aspartate protease-, and aminopeptidase-inhibitors did not inhibit bFGF/FBS-induced S-phase induction in HUVECs, but a proteasome inhibitor, lactacystin did inhibit it reversibly. Lactacystin increased the cellular level of p53 and cdk2-associated p21WAF1/CIP1 leading to cdk2 inactivation. In addition to the angiogenesis inhibitor TNP-470, lactacystin also inhibited the growth of HUVECs selectively at about a 20 times lower concentration than that of other human cell lines, including normal fibroblasts and carcinoma cells. Lactacystin induced p53-dependent p21WAF1/CIP1 expression at lower concentrations in HUVECs than in other cells. These cellular effects were also observed with a tripeptide-type proteasome inhibitor, N-Ac-Leu-Leu-norleucinal.
The proteasome is a protease complex responsible for rapid, selective, and irreversible removal of regulatory proteins, as well as many other cellular proteins. In this study, we have demonstrated that a proliferation-associated nuclear protein Ki-67 depended on the proteasome for its rapid degradation. A proteasome-specific inhibitor lactacystin augmented Ki-67 protein levels in pancreatic cancer BxPC-3 cells while repressed the level of steady-state Ki-67 mRNA. Inhibition of the proteasome also led to accumulation of two CDK inhibitors p27(kip1) and p21(cip1) in the BxPC-3 cells. Failed reduction of Ki-67 protein and enhanced levels of the two CDK inhibitors are likely contributing factors for the suppressed BxPC-3 proliferation after proteasome inhibition.
Multiple Myeloma (MM) is a malignant disease of terminally differentiated B cells. It most likely originates in a B cell which
has traversed the germinal center and has been exposed there extensively to antigens based on the high number of somatic mutations
in the complementarity determining regions. The cell of origin is either a plasmablast, or more likely, a memory B-cell. Typically
MM goes through different phases from indolent (MGUS, smoldering myeloma) to overt myeloma and then to a fulminant phase,
characterized by extramedullary manifestations, high LDH, immature morphology and increased proliferation rate. In the indolent
phase, the disease already has acquired major cytogenetic abnormalities as demonstrated by FISH and DNA flow cytometry. It
has a gene pattern very similar to myeloma cells on gene array analysis. In the early stages of overt MM, the myeloma cells
are completely dependent upon the micro-environment for their growth and survival. The interaction between myeloma cells and
micro-environment causes bone disease, genetic instability and more importantly, drug-resistance, which is caused by upregulation
of anti-apoptotic factors, resistance to apoptosis induced by FAS and TRAIL activation, and by cell adhesion-induced growth
arrest. In this phase of the disease, MM is susceptible to chemotherapy, if delivered with adequate intensity. In the fulminant
phase of MM, myeloma cells have acquired sufficient genetic alternations to become completely independent of the micro-environment
which allows them to grow at extramedullary sites. Because of the many DNA breaks necessary for immature B cells to become
mature plasma cells, B cells already have inherent genetic instability. DNA breaks are necessary for VDJ recombinations, somatic
mutations and isotype switching and it is therefore not surprising that genetic alternations frequently occur at the Ig heavy
chain site at 14q32, which is abnormal in three quarters of myeloma patients. Some of the translocations with 14q32 involve
terminal fragments of chromosomes and can not be diagnosed with standard cytogenetics. Cytogenetic abnormalities are found
in 30–35% of newly diagnosed patients and require sufficient proliferation of MM cells to find enough analyzable mitoses.
The cytogenetic abnormalities are typically complex, involving ≥3 chromosomes in 80% of patients. Almost all chromosomes can
be involved in deletions, additions or translocations of genetic material. Our group has repeatedly stressed the prognostic
significance of chromosome 13 deletion by conventional cytogenetics. The role of chromosome 13 deletion by FISH is less clear.
In addition to chromosome 13 deletion, the presence of a hypodiploid or hypotetraploid karyotye also carries a poor prognosis.
Frequently, deletions of chromosome 13 and hypodiploidy go hand in hand. It remains unclear what specific gene confers the
poor prognosis to patients with deletion 13. The issues of bone disease, drug resistance and cytogenetics will be addressed
in detail during this presentation.
Inhibitors of proteases are currently emerging as a potential anti-cancer modality. Nonselective protease inhibitors are cytotoxic to leukemia and cancer cell lines and we found that this cytotoxicity is correlated with their potency as inhibitors of the proteasome but not as inhibitors of calpain and cathepsin. Highly selective inhibitors of the proteasome were more cytotoxic and fast-acting than less selective inhibitors (PS341>ALLN>ALLM). Induction of wt p53 correlated with inhibition of the proteasome and antiproliferative effect in MCF7, a breast cancer cell line, which was resistant to apoptosis caused by proteasome inhibitors. In contrast, inhibitors of the proteasome induced apoptosis in four leukemia cell lines lacking wt p53. The order of sensitivity of leukemia cells was: Jurkat>HL60> or =U937>K562. The highly selective proteasome inhibitor PS-341 induced cell death with an IC50 as low as 5 nM in apoptosis-prone leukemia cells. Cell death was preceded by p21WAF1/CIP1 accumulation, an alternative marker of proteasome inhibition, and by cleavage of PARP and Rb proteins and nuclear fragmentation. Inhibition of caspases abrogated PARP cleavage and nuclear fragmentation and delayed, but did not completely prevent cell death caused by PS-341. Reintroduction of wt p53 into p53-null PC3 prostate carcinoma cells did not increase their sensitivity to proteasome inhibitors. Likewise, comparison of parental and p21-deficient cells demonstrated that p21WAF1/CIP1 was dispensable for proteasome inhibitor-induced cytotoxicity. We conclude that accumulation of wt p53 and induction of apoptosis are independent markers of proteasome inhibition.
The transcription factor nuclear factor-kappaB (NF-kappaB) regulates genes important for tumor invasion, metastasis and chemoresistance. Normally, NF-kappaB remains sequestered in an inactive state by cytoplasmic inhibitor-of-kappaB (IkappaB) proteins. NF-kappaB translocates to nucleus and activates gene expression upon exposure of cells to growth factors and cytokines. We and others have shown previously that NF-kappaB is constitutively active in a subset of breast cancers. In this study, we show that constitutive activation of NF-kappaB leads to overexpression of the anti-apoptotic genes c-inhibitor of apoptosis 2 (c-IAP2) and manganese superoxide dismutase (Mn-SOD) in breast cancer cells. Furthermore, expression of the anti-apoptotic tumor necrosis factor receptor associated factor 1 (TRAF1) and defender-against cell death (DAD-1) is regulated by NF-kappaB in certain breast cancer cells. We also demonstrate that NF-kappaB-inducible genes protect cancer cells against paclitaxel as MDA-MB-231 breast cancer cells modified to overexpress IkappaBalpha required lower concentrations of paclitaxel to arrest at the G2/M phase of the cell cycle and undergo apoptosis when compared to parental cells. The effect of NF-kappaB on paclitaxel-sensitivity appears to be specific to cancer cells because normal fibroblasts derived from embryos lacking p65 subunit of NF-kappaB and wild type littermate embryos were insensitive to paclitaxel-induced G2/M cell cycle arrest. Parthenolide, an active ingredient of herbal remedies such as feverfew (tanacetum parthenium), mimicked the effects of IkappaBalpha by inhibiting NF-kappaB DNA binding activity and Mn-SOD expression, and increasing paclitaxel-induced apoptosis of breast cancer cells. These results suggest that active ingredients of herbs with anti-inflammatory properties may be useful in increasing the sensitivity of cancers with constitutively active NF-kappaB to chemotherapeutic drugs. Oncogene (2000) 19, 4159 - 4169
Almost all known conventional cytotoxic anticancer drugs are less effective in killing tumor cells grown as multicellular spheroids than in killing tumor cells grown as monolayer cell cultures. This "multicellular resistance" reflects the relative intrinsic drug-resistant phenotype of most solid tumors growing in vivo and is due to factors such as limited drug penetration or reduced fractions of proliferating cells. Proteasome inhibitors such as PS-341, a dipeptide boronic acid analogue, represent an interesting new class of potential anticancer drugs, which are entering early-phase clinical trials. PS-341 has been found to have good broad-spectrum cytotoxic activity in the 60-monolayer cell line National Cancer Institute screen. However, because its relative potency has not been tested in spheroid systems, we analyzed the activity of PS-341 in a spheroid/solid tumor context using four different human ovarian carcinoma cell lines and three prostate carcinoma cell lines, respectively. We found, with one exception, that PS-341 showed equal or greater activity in spheroids than in the respective monolayer cell cultures, even in a prostate cancer spheroid model with a very low growth fraction. PS-341 induced apoptotic cell death in carcinoma cells in both culture systems. We also noted a decrease in XIAP protein, a member of the inhibitor of apoptosis (IAP) family of apoptosis inhibitors, and phosphorylation of Bcl-XL in PS-341-treated ovarian carcinoma cells. Furthermore, DNA fragmentation, a hallmark of apoptosis (in this case, induced by PS-341), was completely inhibited by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD). Taken together, the results indicate that unlike most other known anticancer cytotoxic drugs, PS-341 appears to be as effective in killing tumor cells grown in the form of multicell spheroids as in killing tumor cells grown in monolayer cell culture. Hence, this compound has the potential to circumvent multicellular drug resistance and, as such, may show promising activity against solid tumors with low growth fractions in vivo, which are frequently intrinsically resistant to conventional cytotoxic anticancer drugs.
The 26S proteasome is a non-lysosomal multicatalytic protease complex for degrading intracellular proteins by ATP/ubiquitin-dependent proteolysis. Tightly ordered proteasomal degradation of proteins critical for cell cycle control implies a role of the proteasome in maintaining cell proliferation and cell survival. In this study, we demonstrate that cell-permeable proteasome inhibitors, lactacystin, benzyloxycarbonyl(Z)-leucyl-leucyl-leucinal (ZLLLal; MG-132) and 4-hydroxy-5-iodo-3-nitrophenylacetyl-leucyl-leucyl-leucine vinyl sulfone (NLVS), induce apoptosis abundantly in p53-defective leukemic cell lines CCRF-CEM, U937 and K562 as well as in myelogenic and lymphatic leukemic cells obtained from adult individuals with relapsed acute leukemias. Leukemic cell apoptosis induced by the proteasome inhibitors was dependent on activation of caspase-3 and related caspase family proteases, because caspase-3 inhibitor N-acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspartal (Ac-DEVD-cho) and, more effectively, the general caspase-inhibitor N-benzyloxycarbonyl-L-valyl-L-alanyl-L-aspartate fluoromethylketone (Z-VAD-fmk) were capable of blocking apoptosis induced by lactacystin, ZLLLal or NLVS. Induction of apoptosis by lactacystin or ZLLLal was accompanied by cell cycle arrest at G2/M phase and by accumulation and stabilization of cyclin-dependent kinase inhibitor p21WAF1/Cip and tumor suppressor protein p53. A role of p53 in mediating apoptosis or induction of p21WAF1/Cip1 was ruled out since CCRF-CEM and U937 cells express non-functional mutant p53, and K562 cells lack expression of p53. Viability and hematopoietic outgrowth of human CD34+ progenitor cells treated with lactacystin were slightly reduced, whereas treatment of CD34 + cells with ZLLLal or the cytostatic drugs doxorubicin and gemcitabine resulted in markedly reduced viability and hematopoietic outgrowth. These results demonstrate a basic role of the proteasome in maintaining survival of human leukemic cells, and may define cell-permeable proteasome inhibitors as potently anti-leukemic agents which exhibit a moderate hematopoietic toxicity in vitro.
Human multiple myeloma (MM) is a presently incurable hematological malignancy, and novel biologically based therapies are urgently needed. Proteasome inhibitors represent a novel potential anticancer therapy. In this study, we demonstrate that the proteasome inhibitor PS-341 directly inhibits proliferation and induces apoptosis of human MM cell lines and freshly isolated patient MM cells; inhibits mitogen-activated protein kinase growth signaling in MM cells; induces apoptosis despite induction of p21 and p27 in both p53 wild-type and p53 mutant MM cells; overcomes drug resistance; adds to the anti-MM activity of dexamethasone; and overcomes the resistance to apoptosis in MM cells conferred by interleukin-6. PS-341 also inhibits the paracrine growth of human MM cells by decreasing their adherence to bone marrow stromal cells (BMSCs) and related nuclear factor kappaB-dependent induction of interleukin-6 secretion in BMSCs, as well as inhibiting proliferation and growth signaling of residual adherent MM cells. These data, therefore, demonstrate that PS-341 both acts directly on MM cells and alters cellular interactions and cytokine secretion in the BM millieu to inhibit tumor cell growth, induce apoptosis, and overcome drug resistance. Given the acceptable animal and human toxicity profile of PS-341, these studies provide the framework for clinical evaluation of PS-341 to improve outcome for patients with this universally fatal hematological malignancy.
NF-kappaB is activated by tumor necrosis factor, certain chemotherapeutic agents, and ionizing radiation, leading to inhibition of apoptosis. NF-kappaB activation is regulated by phosphorylation of IkappaB inhibitor molecules that are subsequently targeted for degradation by the ubiquitin-proteasome pathway. PS-341 is a specific and selective inhibitor of the proteasome that inhibits NF-kappaB activation and enhances cytotoxic effects of chemotherapy in vitro and in vivo. The objective of this study was to determine if proteasome inhibition leads to enhanced radiation sensitivity.
Inhibition of NF-kappaB activation in colorectal cancer cells was performed by treatment of LOVO cells with PS-341 or infection with an adenovirus encoding IkappaB super-repressor, a selective NF-kappaB inhibitor. Cells were irradiated at 0, 2, 4, 6, 8, and 10 Gy with or without inhibition of NF-kappaB. NF-kappaB activation was determined by electrophoretic mobility gel shift assay, and apoptosis was evaluated using the TUNEL assay. Growth and clonogenic survival data were obtained to assess effects of treatment on radiosensitization. In vitro results were tested in vivo using a LOVO xenograft model.
NF-kappaB activation was induced by radiation and inhibited by pretreatment with either PS-341 or IkappaBalpha super-repressor in all cell lines. Inhibition of radiation-induced NF-kappaB activation resulted in increased apoptosis and decreased cell growth and clonogenic survival. A 7-41% increase in radiosensitivity was observed for cells treated with PS-341 or IkappaBalpha. An 84% reduction in initial tumor volume was obtained in LOVO xenografts receiving radiation and PS-341.
Inhibition of NF-kappaB activation increases radiation-induced apoptosis and enhances radiosensitivity in colorectal cancer cells in vitro and in vivo. Results are encouraging for the use of PS-341 as a radiosensitizing agent in the treatment of colorectal cancer.
The conjugation of ubiquitin to other cellular proteins regulates a broad range of eukaryotic cell functions. The high efficiency and exquisite selectivity of ubiquitination reactions reflect the properties of enzymes known as ubiquitin-protein ligases or E3s. An E3 recognizes its substrates based on the presence of a specific ubiquitination signal, and catalyzes the formation of an isopeptide bond between a substrate (or ubiquitin) lysine residue and the C terminus of ubiquitin. Although a great deal is known about the molecular basis of E3 specificity, much less is known about molecular mechanisms of catalysis by E3s. Recent findings reveal that all known E3s utilize one of just two catalytic domains--a HECT domain or a RING finger--and crystal structures have provided the first detailed views of an active site of each type. The new findings shed light on many aspects of E3 structure, function, and mechanism, but also emphasize that key features of E3 catalysis remain to be elucidated.
The 26S proteasome degrades proteins that regulate transcription factor activation, cell cycle progression, and apoptosis. In cancer, this may allow for uncontrolled cell division, promoting tumor growth, and spread. We examined whether selective inhibition of the 26S proteasome with PS-341, a dipeptide boronic acid analogue, would block proliferation and induce apoptosis in human pancreatic cancer. Proteasome inhibition significantly blocked mitogen (FCS) induced proliferation of BxPC3 human pancreatic cancer cells in vitro, while arresting cell cycle progression and inducing apoptosis by 24 h. Accumulation of p21(Cip1-Waf-1), a cyclin dependent kinase (CDK) inhibitor normally degraded by the 26S proteasome, occurred by 3 h and correlated with cell cycle arrest. When BxPC3 pancreatic cancer xenografts were established in athymic nu/nu mice, weekly administration of 1 mg/kg PS-341 significantly inhibited tumor growth. Both cellular apoptosis and p21(Cip1-Waf-1) protein levels were increased in PS-341 treated xenografts. Inhibition of tumor xenograft growth was greatest (89%) when PS-341 was combined with the tumoricidal agent CPT-11. Combined CPT-11/PS-341 therapy, but not single agent therapy, yielded highly apoptotic tumors, significantly inhibited tumor cell proliferation, and blocked NF-kappaB activation indicating this systemic therapy was effective at the cancer cell level. 26S proteasome inhibition may represent a new therapeutic approach against this highly resistant and lethal malignancy.
We describe the inhibitory effect of the proteasome inhibitor, lactacystin, on cathepsin A activity in murine melanoma cell lines. In vitro lactacystin metabolite, beta-lactone, at a concentration of 1 microM, significantly suppressed cathepsin A activity in B78 melanoma cell lysates by about 50%. Exposure of three murine melanoma cell lines with different metastatic potential to lactacystin at a concentration of 5 microM for 6 h caused a significant reduction in the carboxypeptidase activity of this enzyme, while the inhibitory activity remained unchanged for at least 12 h. Other proteasome-specific inhibitors, e.g. epoxomicin and N-benzyloxycarbonyl-Ile-Glu(O-tert-Bu)-Ala-leucinal (PSI) at a concentration of 1 microM did not affect cathepsin A activity in melanoma cell line lysates. These data support our previous proposal that lactacystin is not a specific inhibitor of the proteasome. Since cathepsin A is also a tumor-associated enzyme, further research is needed to clarify its role and the significance of its inhibition by lactacystin in tumor biology.
Paclitaxel is a chemotherapeutic drug that induces apoptosis in tumor cells by stabilizing microtubules, prevents normal mitosis, and blocks the cell cycle at the G2/M phase. We have previously reported that the activation of caspase-3 and caspase-8 plays a crucial role in paclitaxel-induced apoptosis. Anti-tumor reagents including paclitaxel, irradiation, and other stimuli activate the transcription factor NF-kappaB, which has the ability to suppress the apoptotic potential of those stimuli. Using a human lung adenocarcinoma cell line (LC-2-AD), we therefore examined whether the inhibition of NF-kappaB activity by proteasome inhibitor 1 (PS1) could become a new adjuvant therapy for cancer. A synergistic effect on apoptosis induction was observed with the combination of more than 0.1 microg/ml paclitaxel and 0.5 microM PS1. An increase in the cell number of apoptotic cells is correlated with the loss of Deltaphim and the activation of caspase-3 and caspase-8. Furthermore, augmented apoptosis is related to NF-kappaB activation. Based on these findings, we propose that the combination of paclitaxel with PS1 could be a new strategy for cancer treatment.
The ubiquitin-proteasome pathway plays a critical role in the degradation of cellular proteins and cell cycle control. Dysregulating the degradation of such proteins should have profound effects on tumor growth and causes cells to undergo apoptosis. The aims of this study are to evaluate the ubiquitin-proteasome pathway in gastric cancer and the potential role of pharmacological inhibition of proteasome on induction of apoptosis in gastric cancer cells. Gastric cancer cell lines AGS (p53 wild-type) and MKN-28 (p53 mutant) were treated with proteasome inhibitor MG132. The results showed that MG132 inhibited cell proliferation in AGS and MKN-28 cells in a time- and dose-dependent manner. The inhibition of cell proliferation was caused by apoptosis which was also time- and dose-dependent. AGS cells were more responsive to MG132 than MKN-28 cells. Induction of apoptosis was preceded by the activation of caspase-3, as measured by a colorimetric caspase-3 cellular activity and Western blotting of the cleavage of caspase-3 and its substrate PARP. Activation of caspase-7 was also exhibited. In addition, z-VAD-fmk, a broad spectrum caspase inhibitor, reversed apoptosis induced by MG132 in AGS and MKN28 cells. Although z-DEVD-fmk, a specific caspase-3 inhibitor, suppressed MG132-induced apoptosis in MKN28 cells, it only partially rescued the apoptotic effect in AGS cells. Caspase-3 activation was the result of release of cytochrome c from mitochondria into the cytosol, as a consequence of upregulation of bax. There were overexpressions of all the proteasome-related proteins p53, p21(waf1) and p27(kip1) at 4 hr after proteasome inhibition which was identified by the accumulation of ubiquitin-tagged proteins. This was accompanied by accumulation of cells at G(1) phase. Our present study suggests that inhibition of proteasome function in gastric cancer cells induces apoptosis and proteasomal inhibitors have potential use as novel anticancer drugs in gastric cancer.
The 20 S proteasome core particle (CP), a multicatalytic protease, is involved in a variety of biologically important processes, including immune response, cell-cycle control, metabolic adaptation, stress response and cell differentiation. Therefore, selective inhibition of the CP will be one possible way to influence these essential pathways. Recently, a new class of specific proteasome inhibitors, TMC-95s, was investigated and we now present a biochemical and crystallographic characterisation of the yeast proteasome core particle in complex with the natural product TMC-95A. This unusual heterocyclic compound specifically blocks the active sites of CPs non-covalently, without modifying the nucleophilic Thr1 residue. The inhibitor is bound to the CP by specific hydrogen bonds with the main-chain atoms of the protein. Analysis of the crystal structure of the complex has revealed which portions of TMC-95s are essential for binding to the proteasome. This will form the basis for the development of synthetic selective proteasome inhibitors as promising candidates for anti-tumoral or anti-inflammatory drugs.
The ubiquitin–proteasome pathway is the major proteolytic system in the cytosol and nucleus of all eukaryotic cells. This ATP-dependent pathway was discovered more than 20 years ago [1] and [2], but the involvement of the proteasome particle was demonstrated only in the late 1980s [3]. Much of our initial understanding of the importance of this pathway in the regulation of different cellular processes came from biochemical studies in extracts of mammalian cells and genetic studies in yeast (reviewed in [4]). However, knowledge about its physiological roles in mammalian cells was slow to develop until cell-permeable proteasome inhibitors were developed, which greatly simplified such studies. Introduction of proteasome inhibitors led to the demonstration that the proteasome catalyzes the degradation not only of the majority of short-lived but also of long-lived proteins, which comprise the bulk of proteins in mammalian cells [5] and [6]. Studies using these compounds have demonstrated that the ubiquitin–proteasome pathway is responsible for the breakdown of a large variety of cell proteins and is essential for many cellular regulatory mechanisms (Table 1). For example, cell cycle progression is controlled by the proteasomal degradation of cyclins and inhibitors of cyclin-dependent kinases [7], while degradation of transcriptional regulators, such as c-Jun, E2F-1 and β-catenin (see [8] for review) is essential for the regulation of cell growth and gene expression. Similarly, degradation by the proteasome of activated protein kinases, e.g. src and protein kinase C [9] and [10], is critical for the termination of certain signal transduction cascades.
Pancreatic cancer is extremely resistant to the induction of apoptosis by chemotherapies; agents that regulate sensitivity to apoptosis may lead to chemosensitization of pancreatic cancer.
MIA-PaCa-2 human pancreatic cancer cells were treated in vitro with the 26S-proteasome inhibitor PS-341. Levels of the apoptosis-regulating proteins (BCL-2, BAK, and BAX) were determined by Western blotting. The effect of PS-341 on BCL-2 gene transcription was examined using a BCL-2 promoter/luciferase reporter construct. The chemosensitizing effect of PS-341 was determined by measurement of the cytotoxic effect of gemcitabine in the presence of PS-341 (10-1000 nM) using the MTT assay. A corresponding in vivo experiment using tumor xenografts in athymic mice was also performed.
PS-341 decreased BCL-2, without effect on BAX or BAK. The downregulation of BCL-2 by PS-341 appears to be transcriptionally mediated. PS-341 induced apoptosis at high does (1000 nM) and increased the cytotoxicity of gemcitabine at low doses (10-100 nM). Xenograft growth was inhibited 59% by gemcitabine; the addition of PS-341 increased growth inhibition to 75%.
Inhibition of the 26S proteasome disrupts the cellular content of key regulators of cell cycle progression and apoptotic control leading to increased sensitivity to standard chemotherapeutic agents, such as gemcitabine, in pancreatic cancer. Combination therapy may lead to better response rates.
Proteasome inhibitors, including lactacystin and MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal), potently induce apoptosis in leukaemic B cells from patients with B cell chronic lymphocytic leukaemia (B-CLL). This pro-apoptotic effect occurs in cells from patients at all stages of the disease, including those resistant to conventional chemotherapy, suggesting that proteasome inhibitors may be useful for treatment of B-CLL. Following initial inhibition of proteasomal activity, these agents induce mitochondrial cytochrome c release and caspase-dependent apoptosis, involving cleavage/activation of caspases -2, -3, -7, -8 and -9. Pre-treatment with the cell permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe)fluoromethyl ketone (Z-VAD.fmk), did not prevent the release of cytochrome c or partial processing of caspase-9 but prevented activation of effector caspases and the induction of apoptosis. These results suggest that the release of cytochrome c is caspase independent and that caspase-9 is the initiator caspase in proteasome inhibitor-induced apoptosis of B-CLL cells. Activation of B-CLL lysates with dATP results in the formation of an approximately 700 kDa caspase-activating apoptosome complex containing Apaf-1. We describe for the first time the formation of a similar approximately 700 kDa caspase-activating apoptosome complex in B-CLL cells induced to undergo apoptosis by proteasome inhibitors.
Aberrant expression of signal transduction molecules in pathways controlling cell survival, proliferation, death, or differentiation are a common feature of all tumors. The identification of the molecules that are involved allows the development of novel tumor-specific strategies. Not surprisingly, targeting these pathways often also results in radiosensitization. The efficacy of such directed therapies may, however, be limited by the heterogeneity and the multiple mutations that are associated with the cancerous state. A more robust alternative may be to target global mechanisms of cellular control. The ubiquitin/proteasome degradation pathway is one candidate for such therapeutic intervention. This pathway is the main posttranscriptional mechanism that controls levels of many short-lived proteins involved in regulation of cell cycle progression, DNA transcription, DNA repair, and apoptosis. Many of these proteins are involved in various malignancies and/or radiation responses. In recent years, proteasome inhibitors have gained interest as a promising new group of antitumor drugs. PS-341, a reversible inhibitor of proteasome chymotryptic activity, is currently being tested in phase I clinical trials. In this study, we show that proteasome inhibition by PS-341 can alter cellular radiosensitivity in vitro and in vivo, in addition to having direct antitumor effects.