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Sample records for proteasome inhibitor lactacystin

  1. Proteasome inhibitors.

    PubMed

    Teicher, Beverly A; Tomaszewski, Joseph E

    2015-07-01

    Proteasome inhibitors have a 20 year history in cancer therapy. The first proteasome inhibitor, bortezomib (Velcade, PS-341), a break-through multiple myeloma treatment, moved rapidly through development from bench in 1994 to first approval in 2003. Bortezomib is a reversible boronic acid inhibitor of the chymotrypsin-like activity of the proteasome. Next generation proteasome inhibitors include carfilzomib and oprozomib which are irreversible epoxyketone proteasome inhibitors; and ixazomib and delanzomib which are reversible boronic acid proteasome inhibitors. Two proteasome inhibitors, bortezomib and carfilzomib are FDA approved drugs and ixazomib and oprozomib are in late stage clinical trials. All of the agents are potent cytotoxics. The disease focus for all the proteasome inhibitors is multiple myeloma. This focus arose from clinical observations made in bortezomib early clinical trials. Later preclinical studies confirmed that multiple myeloma cells were indeed more sensitive to proteasome inhibitors than other tumor cell types. The discovery and development of the proteasome inhibitor class of anticancer agents has progressed through a classic route of serendipity and scientific investigation. These agents are continuing to have a major impact in their treatment of hematologic malignancies and are beginning to be explored as potential treatment agent for non-cancer indications. PMID:25935605

  2. [Proteasome inhibitor].

    PubMed

    Yagi, Hideo

    2014-06-01

    The ubiquitin-proteasome system plays an essential role in degradation of eukaryotic intracellular protein, including cell cycle regulation, cell growth and proliferation, and survival. Cancer cells generally have higher level of proteasome activity compared with normal cells, suggesting proteasome inhibition could be therapeutic target in oncology. Bortezomib, the first proteasome inhibitor introduced into the clinic, is approved for the treatment of patients with multiple myeloma (MM). Although it was approved as single agent in the relapsed setting, bortezomib is now predominantly used in combination with conventional and novel targeted agents because bortezomib has demonstrated additive and synergistic activity in preclinical studies. Recently, several second-generation proteasome inhibitors, such as carfilzomib and MLN9708, have been developed and entered into clinical trials. These agents were investigated in frontline MM in combination with lenalidomide and low-dose dexamethasone. These studies demonstrated positive efficacy and safety, and it is expected that they will be approved in near future. PMID:25016815

  3. Neurorestoration induced by the HDAC inhibitor sodium valproate in the lactacystin model of Parkinson’s is associated with histone acetylation and up-regulation of neurotrophic factors

    PubMed Central

    Harrison, Ian F; Crum, William R; Vernon, Anthony C; Dexter, David T

    2015-01-01

    Background and Purpose Histone hypoacetylation is associated with Parkinson's disease (PD), due possibly to an imbalance in the activities of enzymes responsible for histone (de)acetylation; correction of which may be neuroprotective/neurorestorative. This hypothesis was tested using the anti-epileptic drug sodium valproate, a known histone deacetylase inhibitor (HDACI), utilizing a delayed-start study design in the lactacystin rat model of PD. Experimental Approach The irreversible proteasome inhibitor lactacystin was unilaterally injected into the substantia nigra of Sprague–Dawley rats that subsequently received valproate for 28 days starting 7 days after lactacystin lesioning. Longitudinal motor behavioural testing, structural MRI and post-mortem assessment of nigrostriatal integrity were used to track changes in this model of PD and quantify neuroprotection/restoration. Subsequent cellular and molecular analyses were performed to elucidate the mechanisms underlying valproate's effects. Key Results Despite producing a distinct pattern of structural re-modelling in the healthy and lactacystin-lesioned brain, delayed-start valproate administration induced dose-dependent neuroprotection/restoration against lactacystin neurotoxicity, characterized by motor deficit alleviation, attenuation of morphological brain changes and restoration of dopaminergic neurons in the substantia nigra. Molecular analyses revealed that valproate alleviated lactacystin-induced histone hypoacetylation and induced up-regulation of brain neurotrophic/neuroprotective factors. Conclusions and Implications The histone acetylation and up-regulation of neurotrophic/neuroprotective factors associated with valproate treatment culminate in a neuroprotective and neurorestorative phenotype in this animal model of PD. As valproate induced structural re-modelling of the brain, further research is required to determine whether valproate represents a viable candidate for disease treatment; however

  4. Suppression of cytochrome P450 3A protein levels by proteasome inhibitors.

    SciTech Connect

    Zangar, Richard C. ); Kocarek, Thomas A.; Shen, Shang; Bollinger, Nikki ); Dahn, Michael S.; Lee, Donna W.

    2003-06-01

    We have previously reported that CYP3A cross-links with polyubiquitinated proteins in microsomes from nicardipine-treated rats in a process that is distinct from classical polyubiquitination. To further examine the role of the proteasome in CYP3A degradation, we investigated the effects of proteasome inhibitors lactacystin, MG132, proteasome inhibitor 1, and hemin in primary cultures of rat and human hepatocytes. With the exception of hemin, these agents increased the total pool of ubiquitinated proteins in microsomes isolated from rat hepatocytes, indicating that lactacystin, MG132, and proteasome inhibitor 1 effectively inhibited the proteasome in these cells. All four agents caused a reduction in the amount of the major approximately 55-kDa CYP3A band, opposite to what would be expected if the ubiquitin-proteasome pathway degraded CYP3A. Only hemin treatment caused an increase in high molecular mass (HMM) CYP3A bands. Because hemin treatment did not alter levels of ubiquitin in CYP3 A immunoprecipitates, the HMM CYP3A bands formed in response to hemin treatment clearly were not due to proteasome inhibition. Rather, because hemin treatment also caused an increase in HMM CYP3A in the detergent-insoluble fraction of the 10,000g pellet, the HMM CYP3A seems to represent a large protein complex that is unlikely to primarily represent ubiquitination.

  5. [Proteasome inhibitors in cancer therapy].

    PubMed

    Romaniuk, Wioletta; Ołdziej, Agnieszka Ewa; Zińczuk, Justyna; Kłoczko, Janusz

    2015-01-01

    Proteasomes are multisubunit enzyme complexes. They contain three enzymatic active sites which are termed chymotrypsin-like, trypsin-like, and caspase-like. The elementary function of the proteasomes is degradation of damaged proteins. Proteasome inhibition leads to accumulation of damaged protein, which leads to caspase activation and cell death. This relationship is used in cancer therapy. Bortezomib is the first proteasome inhibitor approved by the US Food and Drug Administration for the treatment of relapsed/refractory multiple myeloma. Carfilzomib belongs to the second generation of drugs, which was approved by the US FDA in 2012. Currently in the study phase there are four new inhibitors: ixazomib (MLN9780/MLN2238), delanzomib (CEP-18770), oprozomib (ONX0912/PR-047) and marizomib (NPI-0052). PMID:27259216

  6. Lactacystin requires reactive oxygen species and Bax redistribution to induce mitochondria-mediated cell death

    PubMed Central

    Perez-Alvarez, Sergio; Solesio, Maria E; Manzanares, Jorge; Jordán, Joaquín; Galindo, María F

    2009-01-01

    Background and purpose: The proteasome inhibitor model of Parkinson's disease (PD) appears to reproduce many of the important behavioural, imaging, pathological and biochemical features of the human disease. However, the mechanisms involved in the lactacystin-induced, mitochondria-mediated apoptotic pathway remain poorly defined. Experimental approach: We have used lactacystin as a specific inhibitor of the 20S proteasome in the dopaminergic neuroblastoma cell line SH-SY5Y. We over-expressed a green fluorescent protein (GFP)–Bax fusion protein in these cells to study localization of Bax. Free radical scavengers were used to assess the role of reactive oxygen species (ROS) in these pathways. Key results: Lactacystin triggered a concentration-dependent increase in cell death mediated by the mitochondrial apoptotic pathway, and induced a change in mitochondrial membrane permeability accompanied by cytochrome c release. The participation of Bax protein was more critical than the formation of the permeability transition pore in mitochondria. GFP–Bax over-expression demonstrated Bax redistribution from the cytosol to mitochondria after the addition of lactacystin. ROS, but not p38 mitogen-activated protein kinase, participated in lactacystin-induced mitochondrial Bax translocation. Lactacystin disrupted the intracellular redox state by increasing ROS production and depleting endogenous antioxidant systems such as glutathione (GSH). Pharmacological depletion of GSH, using l-buthionine sulphoxide, potentiated lactacystin-induced cell death. Lactacystin sensitized neuroblastoma cells to oxidative damage, induced by subtoxic concentrations of 6-hydroxydopamine. Conclusions and implications: The lactacystin-induced, mitochondrial-mediated apoptotic pathway involved interactions between ROS, GSH and Bax. Lactacystin could constitute a potential factor in the development of sporadic PD. PMID:19785649

  7. New proteasome inhibitors in myeloma.

    PubMed

    Lawasut, Panisinee; Chauhan, Dharminder; Laubach, Jacob; Hayes, Catriona; Fabre, Claire; Maglio, Michelle; Mitsiades, Constantine; Hideshima, Teru; Anderson, Kenneth C; Richardson, Paul G

    2012-12-01

    Proteasome inhibition has a validated role in cancer therapy since the successful introduction of bortezomib for the treatment of multiple myeloma (MM) and mantle cell lymphoma, leading to the development of second-generation proteasome inhibitors (PI) for MM patients in whom currently approved therapies have failed. Five PIs have reached clinical evaluation, with the goals of improving efficacy and limiting toxicity, including peripheral neuropathy (PN). Carfilzomib, an epoxyketone with specific chymothrypsin-like activity, acts as an irreversible inhibitor and was recently FDA approved for the response benefit seen in relapsed and refractory MM patients previously treated with bortezomib, thalidomide and lenalidomide. ONX-0912 is now under evaluation as an oral form with similar activity. The boronate peptides MLN9708 and CEP-18770 are orally bioactive bortezomib analogs with prolonged activity and greater tissue penetration. NPI-0052 (marizomib) is a unique, beta-lactone non-selective PI that has been shown to potently overcome bortezomib resistance in vitro. All of these second-generation PIs demonstrate encouraging anti-MM activity and appear to reduce the incidence of PN, with clinical trials ongoing. PMID:23065395

  8. The therapeutic potential of microbial proteasome inhibitors.

    PubMed

    Momose, Isao; Kawada, Manabu

    2016-08-01

    The proteasome influences cellular homeostasis through the degradation of regulatory proteins, many of which are also involved in disease pathogenesis. In particular, numerous regulatory proteins associated with tumor growth, such as cyclins, cyclin-dependent kinase inhibitors, tumor suppressors, and NF-κB inhibitors are degraded by the proteasome. Proteasome inhibitors can stabilize these regulatory proteins, resulting in the suppression of tumor development and the regulation of immune responses. Thus, proteasome inhibitors are promising candidate antitumor agents and immune-regulatory agents. Bortezomib is the first-in-class proteasome inhibitor approved for the treatment of multiple myeloma. Despite its high efficiency, however, a large proportion of patients do not attain sufficient clinical response due to toxicity and drug resistance. Therefore, the development of new proteasome inhibitors with improved pharmacological properties is needed. Natural products produced by microorganisms are a promising source of such compounds. This review provides an overview of proteasome inhibitors produced by microorganisms, with special focus on inhibitors isolated from actinomycetes. PMID:26589840

  9. Proteasome inhibitors induce apoptosis and reduce viral replication in primary effusion lymphoma cells

    SciTech Connect

    Saji, Chiaki; Higashi, Chizuka; Niinaka, Yasufumi; Yamada, Koji; Noguchi, Kohji; Fujimuro, Masahiro

    2011-12-02

    Highlights: Black-Right-Pointing-Pointer Constitutive NF-{kappa}B signaling is essential for the survival and growth of PEL cells. Black-Right-Pointing-Pointer NF-{kappa}B signaling is upregulated by the proteasome-dependent degradation of I{kappa}B{alpha}. Black-Right-Pointing-Pointer Proteasome inhibitors suppress NF-{kappa}B signaling and induce apoptosis in PEL cells through stabilization of I{kappa}B{alpha}. Black-Right-Pointing-Pointer Proteasome inhibitors suppress viral replication in PEL cells during lytic KSHV infection. -- Abstract: Primary effusion lymphoma (PEL) is an aggressive neoplasm caused by Kaposi's sarcoma-associated herpesvirus (KSHV). This study provides evidence that proteasomal activity is required for both survival of PEL cells stably harboring the KSHV genome and viral replication of KSHV. We evaluated the cytotoxic effects of proteasome inhibitors on PEL cells. The proteasome inhibitors MG132, lactacystin, and proteasome inhibitor I dramatically inhibited cell proliferation and induced apoptosis of PEL cells through the accumulation of p21 and p27. Furthermore, proteasome inhibitors induced the stabilization of NF-{kappa}B inhibitory molecule (I{kappa}B{alpha}) and suppressed the transcriptional activity of NF-{kappa}B in PEL cells. The NF-{kappa}B specific inhibitor BAY11-7082 also induced apoptosis in PEL cells. The constitutive activation of NF-{kappa}B signaling is essential for the survival and growth of B cell lymphoma cells, including PEL cells. NF-{kappa}B signaling is upregulated by proteasome-dependent degradation of I{kappa}B{alpha}. The suppression of NF-{kappa}B signaling by proteasome inhibitors may contribute to the induction of apoptosis in PEL cells. In addition, proteasome activity is required for KSHV replication in KSHV latently infected PEL cells. MG132 reduced the production of progeny virus from PEL cells at low concentrations, which do not affect PEL cell growth. These findings suggest that proteasome inhibitors

  10. Proteasome inhibitors remarkably prevent translesion replication in cancer cells but not normal cells.

    PubMed

    Takezawa, Jun; Ishimi, Yukio; Yamada, Kouichi

    2008-05-01

    When a replicative DNA polymerase encounters a lesion on the template strand and stalls, it is replaced with another polymerase(s) with low processivity that bypasses the lesion to continue DNA synthesis. This phenomenon is known as translesion replication or replicative bypass. Failing this, the cell is increasingly likely to undergo apoptosis. In this study, we found that proteasome inhibitors prevent translesion replication in human cancer cells but not in normal cells. Three proteasome inhibitors, MG-132, lactacystin, and MG-262, inhibited UV-induced translesion replication in a wide range of cancer cell lines, including HeLa, HGC-27, MCF-7, HepG2, WiDr, a malignant melanoma, an acute lymphoblastic leukemia, and a multiple myeloma cell line; irrespective of cell origin, histological type, or p53 status. In contrast, these inhibitors had little or no influence on normal fibroblasts (NB1RGB and TIG-1) or a normal liver mesenchymal (LI90) cell line. Among the DNA-damaging antineoplastic agents, cisplatin caused a UV-type translesion reaction; the proteasome inhibitors delayed cisplatin-induced translesion replication in cancer cell lines but had only a weak effect on normal cell lines. Therefore, translesion replication would be an effective target of proteasome inhibitors for cancer chemotherapy by which cancer cells can be efficiently sensitized to DNA-damaging antineoplastic agents, such as cisplatin. PMID:18294277

  11. Substituted quinolines as noncovalent proteasome inhibitors.

    PubMed

    McDaniel, Tanner J; Lansdell, Theresa A; Dissanayake, Amila A; Azevedo, Lauren M; Claes, Jacob; Odom, Aaron L; Tepe, Jetze J

    2016-06-01

    Screening of a library of diverse heterocyclic scaffolds identified substituted quinolines as inhibitors of the human proteasome. The heterocyclic library was prepared via a novel titanium-catalyzed multicomponent coupling reaction, which rendered a diverse set of isoxazoles, pyrimidines, pyrroles, pyrazoles and quinolines. SAR of the parent lead compound indicated that hydrophobic residues on the benzo-moiety significantly improved potency. Lead compound 25 inhibits the chymotryptic-like proteolytic activity of the proteasome (IC50 5.4μM), representing a new class of nonpeptidic, noncovalent proteasome inhibitors. PMID:27112450

  12. Inhibitors Selective for Mycobacterial Versus Human Proteasomes

    SciTech Connect

    Lin, G.; Li, D; Sorio de Carvalho, L; Deng, H; Tao, H; Vogt, G; Wu, K; Schneider, J; Chidawanyika, T; et. al.

    2009-01-01

    Many anti-infectives inhibit the synthesis of bacterial proteins, but none selectively inhibits their degradation. Most anti-infectives kill replicating pathogens, but few preferentially kill pathogens that have been forced into a non-replicating state by conditions in the host. To explore these alternative approaches we sought selective inhibitors of the proteasome of Mycobacterium tuberculosis. Given that the proteasome structure is extensively conserved, it is not surprising that inhibitors of all chemical classes tested have blocked both eukaryotic and prokaryotic proteasomes, and no inhibitor has proved substantially more potent on proteasomes of pathogens than of their hosts. Here we show that certain oxathiazol-2-one compounds kill non-replicating M.?tuberculosis and act as selective suicide-substrate inhibitors of the M.?tuberculosis proteasome by cyclocarbonylating its active site threonine. Major conformational changes protect the inhibitor-enzyme intermediate from hydrolysis, allowing formation of an oxazolidin-2-one and preventing regeneration of active protease. Residues outside the active site whose hydrogen bonds stabilize the critical loop before and after it moves are extensively non-conserved. This may account for the ability of oxathiazol-2-one compounds to inhibit the mycobacterial proteasome potently and irreversibly while largely sparing the human homologue.

  13. Non-invasive evaluation of nigrostriatal neuropathology in a proteasome inhibitor rodent model of Parkinson's disease

    PubMed Central

    2010-01-01

    Background Predominantly, magnetic resonance imaging (MRI) studies in animal models of Parkinson's disease (PD) have focused on alterations in T2 water 1H relaxation or 1H MR spectroscopy (MRS), whilst potential morphological changes and their relationship to histological or behavioural outcomes have not been appropriately addressed. Therefore, in this study we have utilised MRI to scan in vivo brains from rodents bearing a nigrostriatal lesion induced by intranigral injection of the proteasome inhibitor lactacystin. Results Lactacystin induced parkinsonian-like behaviour, characterised by impaired contralateral forelimb grip strength and increased contralateral circling in response to apomorphine. T2-weighted MRI, 3-weeks post-lesion, revealed significant morphological changes in PD-relevant brain areas, including the striatum and ventral midbrain in addition to a decrease in T2 water 1H relaxation in the substantia nigra (SN), but not the striatum. Post-mortem histological analyses revealed extensive dopaminergic neuronal degeneration and α-synuclein aggregation in the SN. However, extensive neuronal loss could also be observed in extra-nigral areas, suggesting non-specific toxicity of lactacystin. Iron accumulation could also be observed throughout the midbrain reflecting changes in T2. Importantly, morphological, but not T2 relaxivity changes, were significantly associated with both behavioural and histological outcomes in this model. Conclusions A pattern of morphological changes in lactacystin-lesioned animals has been identified, as well as alterations in nigral T2 relaxivity. The significant relationship of morphological changes with behavioural and histological outcomes in this model raises the possibility that these may be useful non-invasive surrogate markers of nigrostriatal degeneration in vivo. PMID:20051106

  14. Proteasome inhibitor associated thrombotic microangiopathy.

    PubMed

    Yui, Jennifer C; Van Keer, Jan; Weiss, Brendan M; Waxman, Adam J; Palmer, Matthew B; D'Agati, Vivette D; Kastritis, Efstathios; Dimopoulos, Meletios A; Vij, Ravi; Bansal, Dhruv; Dingli, David; Nasr, Samih H; Leung, Nelson

    2016-09-01

    A variety of medications have been implicated in the causation of thrombotic microangiopathy (TMA). Recently, a few case reports have emerged of TMA attributed to the proteasome inhibitors (PI) bortezomib and carfilzomib in patients with multiple myeloma. The aim of this case series was to better characterize the role of PI in the etiology of drug-induced TMA. We describe eleven patients from six medical centers from around the world who developed TMA while being treated with PI. The median time between medication initiation and diagnosis of TMA was 21 days (range 5 days to 17 months). Median laboratory values at diagnosis included hemoglobin-7.5 g dL(-1) , platelet count-20 × 10(9) /L, LDH-698 U L(-1) , creatinine-3.12 mg dL(-1) . No patient had any other cause of TMA, including ADAMTS13 inhibition, other malignancy or use of any other medication previously associated with TMA. Nine patients had resolution of TMA without evidence of hemolysis after withdrawal of PI. Two patients had stabilization of laboratory values but persistent evidence of hemolysis despite medication withdrawal. One patient had recurrence of TMA with rechallenge of PI. There is a strong level of evidence that PI can cause DITMA. In evaluating patients with suspected TMA, PI use should be recognized as a potential etiology, and these medications should be discontinued promptly if thought to be the cause of TMA. Am. J. Hematol. 91:E348-E352, 2016. © 2016 Wiley Periodicals, Inc. PMID:27286661

  15. Serendipity in discovery of proteasome inhibitors.

    PubMed

    Dunn, Derek; Iqbal, Mohamed; Husten, Jean; Ator, Mark A; Chatterjee, Sankar

    2012-05-15

    Among its various catalytic activities, the 'chymotrypsin-like' activity of the proteasome, a large multicatalytic proteinase complex has emerged as the focus of drug discovery efforts in cancer therapy. Herein, a series of first generation (2S, 3R)-2-amino-3-hydroxybutyric acid derived proteasome inhibitors that were discovered serendipitously en route to original goal of generating a series of sterically constrained oxazoline derivatives has been reported. PMID:22503349

  16. Morphological Changes within the Rat Lateral Ventricle after the Administration of Proteasome Inhibitors.

    PubMed

    Wójcik, Sławomir; Spodnik, Jan Henryk; Dziewiątkowski, Jerzy; Spodnik, Edyta; Moryś, Janusz

    2015-01-01

    The broad variety of substances that inhibit the action of the ubiquitin-proteasome system (UPS)-known as proteasome inhibitors-have been used extensively in previous studies, and they are currently frequently proposed as a novel form of cancer treatment and as a protective factor in intracerebral hemorrhage treatment. The experimental data on the safest route of proteasome inhibitor administration, their associated side effects, and the possible ways of minimizing these effects have recently become a very important topic. The aim of our present study was to determine the effects of administering of MG-132, lactacystin and epoxomicin, compounds belonging to three different classes of proteasome inhibitors, on the ependymal walls of the lateral ventricle. Observations were made 2 and 8 weeks after the intraventricular administration of the studied substances dissolved in dimethyl sulfoxide (DMSO) into the lateral ventricle of adult Wistar rats. Qualitative and quantitative analysis of brain sections stained with histochemical and inmmunofluorescence techniques showed that the administration of proteasome inhibitors caused a partial occlusion of the injected ventricle in all of the studied animals. The occlusion was due to ependymal cells damage and subsequent ependymal discontinuity, which caused direct contact between the striatum and the lateral nuclei of the septum, mononuclear cell infiltration and the formation of a glial scar between these structures (with the activation of astroglia, microglia and oligodendroglia). Morphologically, the ubiquitin-positive aggregates corresponded to aggresomes, indicating impaired activity of the UPS and the accumulation and aggregation of ubiquitinated proteins that coincided with the occurrence of glial scars. The most significant changes were observed in the wall covering the striatum in animals that were administered epoxomicin, and milder changes were observed in animals administered lactacystin and MG-132. Interestingly

  17. Trial Watch: Proteasomal inhibitors for anticancer therapy.

    PubMed

    Obrist, Florine; Manic, Gwenola; Kroemer, Guido; Vitale, Ilio; Galluzzi, Lorenzo

    2015-01-01

    The so-called "ubiquitin-proteasome system" (UPS) is a multicomponent molecular apparatus that catalyzes the covalent attachment of several copies of the small protein ubiquitin to other proteins that are generally (but not always) destined to proteasomal degradation. This enzymatic cascade is crucial for the maintenance of intracellular protein homeostasis (both in physiological conditions and in the course of adaptive stress responses), and regulates a wide array of signaling pathways. In line with this notion, defects in the UPS have been associated with aging as well as with several pathological conditions including cardiac, neurodegenerative, and neoplastic disorders. As transformed cells often experience a constant state of stress (as a result of the hyperactivation of oncogenic signaling pathways and/or adverse microenvironmental conditions), their survival and proliferation are highly dependent on the integrity of the UPS. This rationale has driven an intense wave of preclinical and clinical investigation culminating in 2003 with the approval of the proteasomal inhibitor bortezomib by the US Food and Drug Administration for use in multiple myeloma patients. Another proteasomal inhibitor, carfilzomib, is now licensed by international regulatory agencies for use in multiple myeloma patients, and the approved indications for bortezomib have been extended to mantle cell lymphoma. This said, the clinical activity of bortezomib and carfilzomib is often limited by off-target effects, innate/acquired resistance, and the absence of validated predictive biomarkers. Moreover, the antineoplastic activity of proteasome inhibitors against solid tumors is poor. In this Trial Watch we discuss the contribution of the UPS to oncogenesis and tumor progression and summarize the design and/or results of recent clinical studies evaluating the therapeutic profile of proteasome inhibitors in cancer patients. PMID:27308423

  18. Trial Watch: Proteasomal inhibitors for anticancer therapy

    PubMed Central

    Obrist, Florine; Manic, Gwenola; Kroemer, Guido; Vitale, Ilio; Galluzzi, Lorenzo

    2015-01-01

    The so-called “ubiquitin-proteasome system” (UPS) is a multicomponent molecular apparatus that catalyzes the covalent attachment of several copies of the small protein ubiquitin to other proteins that are generally (but not always) destined to proteasomal degradation. This enzymatic cascade is crucial for the maintenance of intracellular protein homeostasis (both in physiological conditions and in the course of adaptive stress responses), and regulates a wide array of signaling pathways. In line with this notion, defects in the UPS have been associated with aging as well as with several pathological conditions including cardiac, neurodegenerative, and neoplastic disorders. As transformed cells often experience a constant state of stress (as a result of the hyperactivation of oncogenic signaling pathways and/or adverse microenvironmental conditions), their survival and proliferation are highly dependent on the integrity of the UPS. This rationale has driven an intense wave of preclinical and clinical investigation culminating in 2003 with the approval of the proteasomal inhibitor bortezomib by the US Food and Drug Administration for use in multiple myeloma patients. Another proteasomal inhibitor, carfilzomib, is now licensed by international regulatory agencies for use in multiple myeloma patients, and the approved indications for bortezomib have been extended to mantle cell lymphoma. This said, the clinical activity of bortezomib and carfilzomib is often limited by off-target effects, innate/acquired resistance, and the absence of validated predictive biomarkers. Moreover, the antineoplastic activity of proteasome inhibitors against solid tumors is poor. In this Trial Watch we discuss the contribution of the UPS to oncogenesis and tumor progression and summarize the design and/or results of recent clinical studies evaluating the therapeutic profile of proteasome inhibitors in cancer patients. PMID:27308423

  19. The ubiquitin proteasome system and efficacy of proteasome inhibitors in diseases.

    PubMed

    Chitra, Selvarajan; Nalini, Ganesan; Rajasekhar, Gopalakrishnan

    2012-06-01

    In eukaryotes the ubiquitin proteasome pathway plays an important role in cellular homeostasis and also it exerts a critical role in regulating a wide variety of cellular pathways, including cell growth and proliferation, apoptosis, DNA repair, transcription and immune response. Defects in these pathways have been implicated in a number of human pathologies. Inhibition of the ubiquitin proteasome pathway by proteasome inhibitors may be a rational therapeutic approach for various diseases, such as cancer and inflammatory diseases. Many of the critical cytokine and chemokine mediators of the progression of rheumatoid arthritis are regulated by nuclear factor kappa B (NF-κB). In peptidoglycan/polysaccharide-induced polyarthritis, proteasome inhibitors limit the overall inflammation, reduce NF-κB activation, decrease cellular adhesion molecule expression, inhibit nitric oxide synthase, attenuate circulating levels of proinflammatory cytokine interleukin-6 and reduce the arthritis index and swelling in the joints of the animals. Since proteasome inhibitors exhibit anti-inflammatory and anti proliferative effects, diseases characterized by both of these processes such as rheumatoid arthritis might also represent clinical opportunities for such drugs. The regulation of the proteasomal complex by proteasome inhibitors also has implications and potential benefits for the treatment of rheumatoid arthritis. This review summarizes the ubiquitin proteasome pathway, the structure of 26S proteasomes and types of proteasome inhibitors, with their actions, and clinical applications of proteasome inhibitors in various diseases. PMID:22709487

  20. Clinical and marketed proteasome inhibitors for cancer treatment.

    PubMed

    Zhang, Jiankang; Wu, Peng; Hu, Yongzhou

    2013-01-01

    The ubiquitin-proteasome pathway (UPP), which influences essential cellular functions including cell growth, differentiation, apoptosis, signal transduction, antigen processing and inflammatory responses, has been considered as one of the most important cellular protein degradation approaches. Proteasome functions as a gatekeeper, which controls the execution of protein degradation and plays a critical role in the ubiquitin-proteasome pathway. The unfolding of the close connection between proteasome and cancer provides a potential strategy for cancer treatment by using proteasome inhibitors. Small molecular inhibitors of varied structures and potency against proteasome have been discovered in recent years, with bortezomib and carfilzomib having been successfully approved for clinical application while some other promising candidates are currently under clinical trials. Herein, we review the development history of drugs and candidates that target the 20S proteasome, structure-activity relationships (SARs) of various proteasome inhibitors, and related completed or ongoing clinical trials. PMID:23531219

  1. Effect of proteasome inhibition on toxicity and CYP3A23 induction in cultured rat hepatocytes: Comparison with arsenite

    SciTech Connect

    Noreault-Conti, Trisha L.; Jacobs, Judith M.; Trask, Heidi W.; Wrighton, Steven A.; Sinclair, Jacqueline F.; Nichols, Ralph C. . E-mail: ralph.c.nichols@dartmouth.edu

    2006-12-15

    Previous work in our laboratory has shown that acute exposure of primary rat hepatocyte cultures to non-toxic concentrations of arsenite causes major decreases in the DEX-mediated induction of CYP3A23 protein, with minor decreases in CYP3A23 mRNA. To elucidate the mechanism for these effects of arsenite, the effects of arsenite and proteasome inhibition, separately and in combination, on induction of CYP3A23 protein were compared. The proteasome inhibitor, MG132, inhibited proteasome activity, but also decreased CYP3A23 mRNA and protein. Lactacystin, another proteasome inhibitor, decreased CYP3A23 protein without affecting CYP3A23 mRNA at a concentration that effectively inhibited proteasome activity. This result, suggesting that the action of lactacystin is similar to arsenite and was post-transcriptional, was confirmed by the finding that lactacystin decreased association of DEX-induced CYP3A23 mRNA with polyribosomes. Both MG132 and lactacystin inhibited total protein synthesis, but did not affect MTT reduction. Arsenite had no effect on ubiquitination of proteins, nor did arsenite significantly affect proteasomal activity. These results suggest that arsenite and lactacystin act by similar mechanisms to inhibit translation of CYP3A23.

  2. Development of novel proteasome inhibitors based on phthalazinone scaffold.

    PubMed

    Yang, Lingfei; Wang, Wei; Sun, Qi; Xu, Fengrong; Niu, Yan; Wang, Chao; Liang, Lei; Xu, Ping

    2016-06-15

    In this study we designed a series of proteasome inhibitors using pyridazinone as initial scaffold, and extended the structure with rational design by computer aided drug design (CADD). Two different synthetic routes were explored and the biological evaluation of the phthalazinone derivatives was investigated. Most importantly, electron positive triphenylphosphine group was first introduced in the structure of proteasome inhibitors and potent inhibition was achieved. As 6c was the most potent inhibitor of proteasome, we examined the structure-activity relationship (SAR) of 6c analogs. PMID:27158142

  3. Overview of Proteasome Inhibitor-Based Anti-cancer Therapies: Perspective on Bortezomib and Second Generation Proteasome Inhibitors versus Future Generation Inhibitors of Ubiquitin-Proteasome System

    PubMed Central

    Dou, Q. Ping; Zonder, Jeffrey A.

    2014-01-01

    Over the past ten years, proteasome inhibition has emerged as an effective therapeutic strategy for treating multiple myeloma (MM) and some lymphomas. In 2003, Bortezomib (BTZ) became the first proteasome inhibitor approved by the U.S. Food and Drug Administration (FDA). BTZ-based therapies have become a staple for the treatment of MM at all stages of the disease. The survival rate of MM patients has improved significantly since clinical introduction of BTZ and other immunomodulatory drugs. However, BTZ has several limitations. Not all patients respond to BTZ-based therapies and relapse occurs in many patients who initially responded. Solid tumors, in particular, are often resistant to BTZ. Furthermore, BTZ can induce dose-limiting peripheral neuropathy (PN). The second generation proteasome inhibitor Carfizomib (CFZ; U.S. FDA approved in August 2012) induces responses in a minority of MM patients relapsed from or refractory to BTZ. There is less PN compared to BTZ. Four other second-generation proteasome inhibitors (Ixazomib, Delanzomib, Oprozomib and Marizomib) with different pharmacologic properties and broader anticancer activities, have also shown some clinical activity in bortezomib-resistant cancers. While the mechanism of resistance to bortezomib in human cancers still remains to be fully understood, targeting the immunoproteasome, ubiquitin E3 ligases, the 19S proteasome and deubiquitinases in pre-clinical studies represents possible directions for future generation inhibitors of ubiquitin-proteasome system in the treatment of MM and other cancers. PMID:25092212

  4. Overview of proteasome inhibitor-based anti-cancer therapies: perspective on bortezomib and second generation proteasome inhibitors versus future generation inhibitors of ubiquitin-proteasome system.

    PubMed

    Dou, Q Ping; Zonder, Jeffrey A

    2014-01-01

    Over the past ten years, proteasome inhibition has emerged as an effective therapeutic strategy for treating multiple myeloma (MM) and some lymphomas. In 2003, Bortezomib (BTZ) became the first proteasome inhibitor approved by the U.S. Food and Drug Administration (FDA). BTZ-based therapies have become a staple for the treatment of MM at all stages of the disease. The survival rate of MM patients has improved significantly since clinical introduction of BTZ and other immunomodulatory drugs. However, BTZ has several limitations. Not all patients respond to BTZ based therapies and relapse occurs in many patients who initially responded. Solid tumors, in particular, are often resistant to BTZ. Furthermore, BTZ can induce dose-limiting peripheral neuropathy (PN). The second generation proteasome inhibitor Carfizomib (CFZ; U.S. FDA approved in August 2012) induces responses in a minority of MM patients relapsed from or refractory to BTZ. There is less PN compared to BTZ. Four other second-generation proteasome inhibitors (Ixazomib, Delanzomib, Oprozomib and Marizomib) with different pharmacologic properties and broader anticancer activities, have also shown some clinical activity in bortezomib-resistant cancers. While the mechanism of resistance to bortezomib in human cancers still remains to be fully understood, targeting the immunoproteasome, ubiquitin E3 ligases, the 19S proteasome and deubiquitinases in pre-clinical studies represents possible directions for future generation inhibitors of ubiquitin-proteasome system in the treatment of MM and other cancers. PMID:25092212

  5. Chronic L-DOPA treatment attenuates behavioral and biochemical deficits induced by unilateral lactacystin administration into the rat substantia nigra.

    PubMed

    Konieczny, Jolanta; Czarnecka, Anna; Lenda, Tomasz; Kamińska, Kinga; Lorenc-Koci, Elżbieta

    2014-03-15

    The aim of the study was to determine whether the dopamine (DA) precursor l-DOPA attenuates parkinsonian-like symptoms produced by the ubiquitin-proteasome system inhibitor lactacystin. Wistar rats were injected unilaterally with lactacystin (2.5 μg/2 μl) or 6-OHDA (8 μg/2 μl) into the substantia nigra (SN) pars compacta. Four weeks after the lesion, the animals were treated chronically with l-DOPA (25 or 50 mg/kg) for two weeks. During l-DOPA treatment, the lactacystin-treated rats were tested for catalepsy and forelimb asymmetry. Rotational behavior was evaluated after apomorphine (0.25 mg/kg) and l-DOPA in both PD models. After completion of experiments, the animals were killed and the levels of DA and its metabolites in the striatum and SN were assayed. We found that acute l-DOPA administration effectively decreased catalepsy and increased the use of the compromised forelimb in the cylinder test. However, the lactacystin group did not respond to apomorphine or acute l-DOPA administration in the rotational test. Repeated l-DOPA treatment produced contralateral rotations in both PD models, but the number of rotations was much greater in the 6-OHDA-lesioned rats. Both toxins markedly (>90%) reduced the levels of DA and its metabolites in the striatum and SN, while l-DOPA diminished these decreases, especially in the SN. By demonstrating the efficacy of l-DOPA in several behavioral tests, our study confirms the usefulness of the lactacystin lesion as a model of PD. However, marked differences in the rotational response to apomorphine and l-DOPA suggest different mechanisms of neurodegeneration evoked by lactacystin and 6-OHDA. PMID:24361083

  6. Proteasome Inhibitors: An Expanding Army Attacking a Unique Target

    PubMed Central

    Kisselev, Alexei F.; van der Linden, Wouter A.; Overkleeft, Herman S.

    2012-01-01

    Proteasomes are large, multisubunit proteolytic complexes presenting multiple targets for therapeutic intervention. The 26S proteasome consists of a 20S proteolytic core and one or two 19S regulatory particles. The 20S core contains three types of active sites. Many structurally diverse inhibitors of these active sites, both natural product and synthetic, have been discovered in the last two decades. One, bortezomib, is used clinically for treatment of multiple myeloma, mantle cell lymphoma, and acute allograft rejection. Five more recently developed proteasome inhibitors are in trials for treatment of myeloma and other cancers. Proteasome inhibitors also have activity in animal models of autoimmune and inflammatory diseases, reperfusion injury, promote bone and hair growth, and can potentially be used as anti-infectives. In addition, inhibitors of ATPases and deubiquitinases of 19S regulatory particles have been discovered in the last decade. PMID:22284358

  7. Identification of Novel Proteasome Inhibitors from an Enaminone Library.

    PubMed

    Elliott, Megan L; Thomas, Kevin; Kennedy, Steven; Koduri, Naga D; Hussaini, R Syed; Sheaff, Robert J

    2015-09-01

    A library of structurally distinct enaminones was synthesized using sonication or Ru(II) catalysis to couple primary, secondary, and tertiary thioamides with α-halocarbonyls or α-diazocarbonyls. Screening the library for proteasome inhibition using a luciferase-based assay identified seven structurally diverse compounds. Two of these molecules targeted luciferase, while the remaining five exhibited varying potency and specificity for the trypsin-like, chymotrypsin-like, or caspase-like protease activities of the proteasome. Physiological relevance was confirmed by showing these molecules inhibited proteasomal degradation of the full-length protein substrate p21cip1 expressed in tissue culture cells. A cell viability analysis revealed that the proteasome inhibitors differentially affected cell survival. Results indicate a subset of enaminones and precursor molecules identified in this study are good candidates for further development into novel proteasome inhibitors with potential therapeutic value. PMID:25494709

  8. From Bortezomib to other Inhibitors of the Proteasome and Beyond

    PubMed Central

    Buac, Daniela; Shen, Min; Schmitt, Sara; Kona, Fathima Rani; Deshmukh, Rahul; Zhang, Zhen; Neslund-Dudas, Christine; Mitra, Bharati; Dou, Q. Ping

    2013-01-01

    The cancer drug discovery field has placed much emphasis on the identification of novel and cancer-specific molecular targets. A rich source of such targets for the design of novel anti-tumor agents is the ubiqutin-proteasome system (UP-S), a tightly regulated, highly specific pathway responsible for the vast majority of protein turnover within the cell. Because of its critical role in almost all cell processes that ensure normal cellular function, its inhibition at one point in time was deemed non-specific and therefore not worth further investigation as a molecular drug target. However, today the proteasome is one of the most promising anti-cancer drug targets of the century. The discovery that tumor cells are in fact more sensitive to proteasome inhibitors than normal cells indeed paved the way for the design of its inhibitors. Such efforts have led to bortezomib, the first FDA approved proteasome inhibitor now used as a frontline treatment for newly diagnosed multiple myeloma (MM), relapsed/refractory MM and mantle cell lymphoma. Though successful in improving clinical outcomes for patients with hematological malignancies, relapse often occurs in those who initially responded to bortezomib. Therefore, the acquisition of bortezomib resistance is a major issue with its therapy. Furthermore, some neuro-toxicities have been associated with bortezomib treatment and its efficacy in solid tumors is lacking. These observations have encouraged researchers to pursue the next generation of proteasome inhibitors, which would ideally overcome bortezomib resistance, have reduced toxicities and a broader range of anti-cancer activity. This review summarizes the success and limitations of bortezomib, and describes recent advances in the field, including, and most notably, the most recent FDA approval of carfilzomib in July, 2012, a second generation proteasome inhibitor. Other proteasome inhibitors currently in clinical trials and those that are currently experimental grade

  9. Rational Design of Proteasome Inhibitors as Antimalarial Drugs.

    PubMed

    Le Chapelain, Camille; Groll, Michael

    2016-05-23

    One life, two strategies: Crucial structural differences between the human and the Plasmodium falciparum proteasomes were recently identified. A combination of cryo-EM and functional characterization enabled the design of a selective antimalarial proteasome inhibitor that shows low toxicity in the host. When used with artemisinin, this ligand offers a new approach for the efficient treatment of malaria at all stages of the parasite lifecycle. PMID:27079849

  10. Limb-girdle muscular dystrophy (LGMD-1C) mutants of caveolin-3 undergo ubiquitination and proteasomal degradation. Treatment with proteasomal inhibitors blocks the dominant negative effect of LGMD-1C mutanta and rescues wild-type caveolin-3.

    PubMed

    Galbiati, F; Volonte, D; Minetti, C; Bregman, D B; Lisanti, M P

    2000-12-01

    Caveolin-3 is the principal structural protein of caveolae in striated muscle. Autosomal dominant limb-girdle muscular dystrophy (LGMD-1C) in humans is due to mutations (DeltaTFT and Pro --> Leu) within the CAV3 gene. We have shown that LGMD-1C mutations lead to formation of unstable aggregates of caveolin-3 that are retained intracellularly and are rapidly degraded. The mechanism by which LGMD-1C mutants of caveolin-3 are degraded remains unknown. Here, we show that LGMD-1C mutants of caveolin-3 undergo ubiquitination-proteasomal degradation. Treatment with proteasomal inhibitors (MG-132, MG-115, lactacystin, or proteasome inhibitor I), but not lysosomal inhibitors, prevented degradation of LGMD-1C caveolin-3 mutants. In the presence of MG-132, LGMD-1C caveolin-3 mutants accumulated within the endoplasmic reticulum and did not reach the plasma membrane. LGMD-1C mutants of caveolin-3 behave in a dominant negative fashion, causing intracellular retention and degradation of wild-type caveolin-3. Interestingly, in cells co-expressing wild-type and mutant forms of caveolin-3, MG-132 treatment rescued wild-type caveolin-3; wild-type caveolin-3 was not degraded and reached the plasma membrane. These results may have clinical implications for treatment of patients with LGMD-1C. PMID:10973975

  11. New orally active proteasome inhibitors in multiple myeloma.

    PubMed

    Allegra, Alessandro; Alonci, Andrea; Gerace, Demetrio; Russo, Sabina; Innao, Vanessa; Calabrò, Laura; Musolino, Caterina

    2014-01-01

    Bortezomib is the first proteasome inhibitor approved for the therapy of multiple myeloma (MM). Although Bortezomib has renovated the treatment of MM, a considerable proportion of subjects fail to respond to Bortezomib treatment and almost all patients relapse from this drug either alone or when used in combination therapies. However, the good clinical outcome of Bortezomib treatment in MM patients gave impulsion for the development of second generation proteasome inhibitors with the ambition of improving efficacy of proteasome inhibition, enhancing antitumor activity, and decreasing toxicity, as well as providing flexible dosing schedules and patient convenience. This review provides an overview of the role of oral proteasome inhibitors including Marizomib, Oprozomib, Delanzomib, chemical proteasome inhibitors, and cinnabaramides, in the therapy of MM, focusing on developments over the past five years. These emerging drugs with different mechanisms of action have exhibited promising antitumor activity in patients with relapsed/refractory MM, and they are creating chances to target multiple pathways, overcome resistance, and improve clinical outcomes, mainly for those subjects who are refractory to approved agents. Future steps in the clinical development of oral inhibitors include the optimization of the schedule and the definition of their antitumor activity in MM. PMID:24239172

  12. Subunit specific inhibitors of proteasomes and their potential for immunomodulation

    PubMed Central

    Kisselev, Alexei F; Groettrup, Marcus

    2015-01-01

    Specialized variants of the constitutive 20S proteasome in the immune system like the immunoproteasomes and the thymoproteasome contain active site-bearing subunits which differ in their cleavage priorities and substrate binding pockets. The immunoproteasome plays a crucial role in antigen processing and for the differentiation of pro-inflammatory T helper cells which are involved in the pathogenesis of autoimmunity. Selective inhibitors of the immunoproteasome and constitutive proteasome have recently been generated which interfere with the development and progression of autoimmune diseases. Here we describe these inhibitors and their therapeutic potential as predicted from preclinical models. PMID:25217863

  13. Proteasome Inhibitors in the Treatment of Multiple Myeloma

    PubMed Central

    Shah, Jatin J.; Orlowski, Robert Z.

    2016-01-01

    Targeting intracellular protein turnover by inhibiting the ubiquitin-proteasome pathway as a strategy for cancer therapy is a new addition to our chemotherapeutic armamentarium, and has seen its greatest successes against multiple myeloma. The first-in-class proteasome inhibitor bortezomib was initially approved for treatment of patients in the relapsed/refractory setting as a single agent, and was recently shown to induce even greater benefits as part of rationally-designed combinations that overcome chemoresistance. Modulation of proteasome function is also a rational approach to achieve chemosensitization to other anti-myeloma agents, and bortezomib has now been incorporated into the front-line setting. Bortezomib-based induction regimens are able to achieve higher overall response rates and response qualities than was the case with prior standards of care, and unlike these older approaches, maintain efficacy in patients with clinically- and molecularly-defined high-risk disease. Second-generation proteasome inhibitors with novel properties, such as NPI-0052 and carfilzomib, are entering the clinical arena, and showing evidence of anti-myeloma activity. In this spotlight review, we provide an overview of the current state of the art use of bortezomib and other proteasome inhibitors against multiple myeloma, and highlight areas for future study that will further optimize our ability to benefit patients with this disease. PMID:19741722

  14. A novel proteasome inhibitor NPI-0052 as an anticancer therapy

    PubMed Central

    Chauhan, D; Hideshima, T; Anderson, K C

    2006-01-01

    Proteasome inhibitor Bortezomib/Velcade has emerged as an effective anticancer therapy for the treatment of relapsed and/or refractory multiple myeloma (MM), but prolonged treatment can be associated with toxicity and development of drug resistance. In this review, we discuss the recent discovery of a novel proteasome inhibitor, NPI-0052, that is distinct from Bortezomib in its chemical structure, mechanisms of action, and effects on proteasomal activities; most importantly, it overcomes resistance to conventional and Bortezomib therapies. In vivo studies using human MM xenografts shows that NPI-0052 is well tolerated, prolongs survival, and reduces tumour recurrence. These preclinical studies provided the basis for Phase-I clinical trial of NPI-0052 in relapsed/refractory MM patients. PMID:17047643

  15. Evolution of Extra-Nigral Damage Predicts Behavioural Deficits in a Rat Proteasome Inhibitor Model of Parkinson's Disease

    PubMed Central

    Vernon, Anthony C.; Crum, William R.; Johansson, Saga M.; Modo, Michel

    2011-01-01

    Establishing the neurological basis of behavioural dysfunction is key to provide a better understanding of Parkinson's disease (PD) and facilitate development of effective novel therapies. For this, the relationships between longitudinal structural brain changes associated with motor behaviour were determined in a rat model of PD and validated by post-mortem immunohistochemistry. Rats bearing a nigrostriatal lesion induced by infusion of the proteasome inhibitor lactacystin into the left-medial forebrain bundle and saline-injected controls underwent magnetic resonance imaging (MRI) at baseline (prior to surgery) and 1, 3 and 5 weeks post-surgery with concomitant motor assessments consisting of forelimb grip strength, accelerating rotarod, and apormorphine-induced rotation. Lactacystin-injected rats developed early motor deficits alongside decreased ipsilateral cortical volumes, specifically thinning of the primary motor (M1) and somatosensory cortices and lateral ventricle hypertrophy (as determined by manual segmentation and deformation-based morphometry). Although sustained, motor dysfunction and nigrostriatal damage were maximal by 1 week post-surgery. Additional volume decreases in the ipsilateral ventral midbrain; corpus striatum and thalamus were only evident by week 3 and 5. Whilst cortical MRI volume changes best predicted the degree of motor impairment, post-mortem tyrosine hydroxylase immunoreactivity in the striatum was a better predictor of motor behaviour overall, with the notable exception of performance in the accelerating rotarod, in which, M1 cortical thickness remained the best predictor. These results highlight the importance of identifying extra-nigral regions of damage that impact on behavioural dysfunction from damage to the nigrostriatal system. PMID:21364887

  16. Proteasome inhibitors - molecular basis and current perspectives in multiple myeloma.

    PubMed

    Kubiczkova, Lenka; Pour, Ludek; Sedlarikova, Lenka; Hajek, Roman; Sevcikova, Sabina

    2014-06-01

    Inhibition of proteasome, a proteolytic complex responsible for the degradation of ubiquitinated proteins, has emerged as a powerful strategy for treatment of multiple myeloma (MM), a plasma cell malignancy. First-in-class agent, bortezomib, has demonstrated great positive therapeutic efficacy in MM, both in pre-clinical and in clinical studies. However, despite its high efficiency, a large proportion of patients do not achieve sufficient clinical response. Therefore, the development of a second-generation of proteasome inhibitors (PIs) with improved pharmacological properties was needed. Recently, several of these new agents have been introduced into clinics including carfilzomib, marizomib and ixazomib. Further, new orally administered second-generation PI oprozomib is being investigated. This review provides an overview of main mechanisms of action of PIs in MM, focusing on the ongoing development and progress of novel anti-proteasome therapeutics. PMID:24712303

  17. Characterisation of 20S Proteasome in Tritrichomonas foetus and Its Role during the Cell Cycle and Transformation into Endoflagellar Form

    PubMed Central

    Pereira-Neves, Antonio; Gonzaga, Luiz; Menna-Barreto, Rubem F. S.; Benchimol, Marlene

    2015-01-01

    Proteasomes are intracellular complexes that control selective protein degradation in organisms ranging from Archaea to higher eukaryotes. These structures have multiple proteolytic activities that are required for cell differentiation, replication and maintaining cellular homeostasis. Here, we document the presence of the 20S proteasome in the protist parasite Tritrichomonas foetus. Complementary techniques, such as a combination of whole genome sequencing technologies, bioinformatics algorithms, cell fractionation and biochemistry and microscopy approaches were used to characterise the 20S proteasome of T. foetus. The 14 homologues of the typical eukaryotic proteasome subunits were identified in the T. foetus genome. Alignment analyses showed that the main regulatory and catalytic domains of the proteasome were conserved in the predicted amino acid sequences from T. foetus-proteasome subunits. Immunofluorescence assays using an anti-proteasome antibody revealed a labelling distributed throughout the cytosol as punctate cytoplasmic structures and in the perinuclear region. Electron microscopy of a T. foetus-proteasome-enriched fraction confirmed the presence of particles that resembled the typical eukaryotic 20S proteasome. Fluorogenic assays using specific peptidyl substrates detected presence of the three typical peptidase activities of eukaryotic proteasomes in T. foetus. As expected, these peptidase activities were inhibited by lactacystin, a well-known specific proteasome inhibitor, and were not affected by inhibitors of serine or cysteine proteases. During the transformation of T. foetus to endoflagellar form (EFF), also known as pseudocyst, we observed correlations between the EFF formation rates, increases in the proteasome activities and reduced levels of ubiquitin-protein conjugates. The growth, cell cycle and EFF transformation of T. foetus were inhibited after treatment with lactacystin in a dose-dependent manner. Lactacystin treatment also resulted in

  18. Characterisation of 20S Proteasome in Tritrichomonas foetus and Its Role during the Cell Cycle and Transformation into Endoflagellar Form.

    PubMed

    Pereira-Neves, Antonio; Gonzaga, Luiz; Menna-Barreto, Rubem F S; Benchimol, Marlene

    2015-01-01

    Proteasomes are intracellular complexes that control selective protein degradation in organisms ranging from Archaea to higher eukaryotes. These structures have multiple proteolytic activities that are required for cell differentiation, replication and maintaining cellular homeostasis. Here, we document the presence of the 20S proteasome in the protist parasite Tritrichomonas foetus. Complementary techniques, such as a combination of whole genome sequencing technologies, bioinformatics algorithms, cell fractionation and biochemistry and microscopy approaches were used to characterise the 20S proteasome of T. foetus. The 14 homologues of the typical eukaryotic proteasome subunits were identified in the T. foetus genome. Alignment analyses showed that the main regulatory and catalytic domains of the proteasome were conserved in the predicted amino acid sequences from T. foetus-proteasome subunits. Immunofluorescence assays using an anti-proteasome antibody revealed a labelling distributed throughout the cytosol as punctate cytoplasmic structures and in the perinuclear region. Electron microscopy of a T. foetus-proteasome-enriched fraction confirmed the presence of particles that resembled the typical eukaryotic 20S proteasome. Fluorogenic assays using specific peptidyl substrates detected presence of the three typical peptidase activities of eukaryotic proteasomes in T. foetus. As expected, these peptidase activities were inhibited by lactacystin, a well-known specific proteasome inhibitor, and were not affected by inhibitors of serine or cysteine proteases. During the transformation of T. foetus to endoflagellar form (EFF), also known as pseudocyst, we observed correlations between the EFF formation rates, increases in the proteasome activities and reduced levels of ubiquitin-protein conjugates. The growth, cell cycle and EFF transformation of T. foetus were inhibited after treatment with lactacystin in a dose-dependent manner. Lactacystin treatment also resulted in

  19. The novel β2-selective proteasome inhibitor LU-102 synergizes with bortezomib and carfilzomib to overcome proteasome inhibitor resistance of myeloma cells.

    PubMed

    Kraus, Marianne; Bader, Juergen; Geurink, Paul P; Weyburne, Emily S; Mirabella, Anne C; Silzle, Tobias; Shabaneh, Tamer B; van der Linden, Wouter A; de Bruin, Gerjan; Haile, Sarah R; van Rooden, Eva; Appenzeller, Christina; Li, Nan; Kisselev, Alexei F; Overkleeft, Herman; Driessen, Christoph

    2015-10-01

    Proteasome inhibitor resistance is a challenge for myeloma therapy. Bortezomib targets the β5 and β1 activity, but not the β2 activity of the proteasome. Bortezomib-resistant myeloma cells down-regulate the activation status of the unfolded protein response, and up-regulate β2 proteasome activity. To improve proteasome inhibition in bortezomib-resistant myeloma and to achieve more efficient UPR activation, we have developed LU-102, a selective inhibitor of the β2 proteasome activity. LU-102 inhibited the β2 activity in intact myeloma cells at low micromolar concentrations without relevant co-inhibition of β1 and β5 proteasome subunits. In proteasome inhibitor-resistant myeloma cells, significantly more potent proteasome inhibition was achieved by bortezomib or carfilzomib in combination with LU-102, compared to bortezomib/carfilzomib alone, resulting in highly synergistic cytotoxic activity of the drug combination via endoplasmatic reticulum stress-induced apoptosis. Combining bortezomib/carfilzomib with LU-102 significantly prolonged proteasome inhibition and increased activation of the unfolded protein response and IRE1-a activity. IRE1-α has recently been shown to control myeloma cell differentiation and bortezomib sensitivity (Leung-Hagesteijn, Cancer Cell 24:3, 289-304). Thus, β2-selective proteasome inhibition by LU-102 in combination with bortezomib or carfilzomib results in synergistic proteasome inhibition, activation of the unfolded protein response, and cytotoxicity, and overcomes bortezomib/carfilzomib resistance in myeloma cells in vitro. PMID:26069288

  20. The novel β2-selective proteasome inhibitor LU-102 synergizes with bortezomib and carfilzomib to overcome proteasome inhibitor resistance of myeloma cells

    PubMed Central

    Kraus, Marianne; Bader, Juergen; Geurink, Paul P.; Weyburne, Emily S.; Mirabella, Anne C.; Silzle, Tobias; Shabaneh, Tamer B.; van der Linden, Wouter A.; de Bruin, Gerjan; Haile, Sarah R.; van Rooden, Eva; Appenzeller, Christina; Li, Nan; Kisselev, Alexei F.; Overkleeft, Herman; Driessen, Christoph

    2015-01-01

    Proteasome inhibitor resistance is a challenge for myeloma therapy. Bortezomib targets the β5 and β1 activity, but not the β2 activity of the proteasome. Bortezomib-resistant myeloma cells down-regulate the activation status of the unfolded protein response, and up-regulate β2 proteasome activity. To improve proteasome inhibition in bortezomib-resistant myeloma and to achieve more efficient UPR activation, we have developed LU-102, a selective inhibitor of the β2 proteasome activity. LU-102 inhibited the β2 activity in intact myeloma cells at low micromolar concentrations without relevant co-inhibition of β1 and β5 proteasome subunits. In proteasome inhibitor-resistant myeloma cells, significantly more potent proteasome inhibition was achieved by bortezomib or carfilzomib in combination with LU-102, compared to bortezomib/carfilzomib alone, resulting in highly synergistic cytotoxic activity of the drug combination via endoplasmatic reticulum stress-induced apoptosis. Combining bortezomib/carfilzomib with LU-102 significantly prolonged proteasome inhibition and increased activation of the unfolded protein response and IRE1-a activity. IRE1-α has recently been shown to control myeloma cell differentiation and bortezomib sensitivity (Leung-Hagesteijn, Cancer Cell 24:3, 289-304). Thus, β2-selective proteasome inhibition by LU-102 in combination with bortezomib or carfilzomib results in synergistic proteasome inhibition, activation of the unfolded protein response, and cytotoxicity, and overcomes bortezomib/carfilzomib resistance in myeloma cells in vitro PMID:26069288

  1. ROS inhibitor N-acetyl-L-cysteine antagonizes the activity of proteasome inhibitors.

    PubMed

    Halasi, Marianna; Wang, Ming; Chavan, Tanmay S; Gaponenko, Vadim; Hay, Nissim; Gartel, Andrei L

    2013-09-01

    NAC (N-acetyl-L-cysteine) is commonly used to identify and test ROS (reactive oxygen species) inducers, and to inhibit ROS. In the present study, we identified inhibition of proteasome inhibitors as a novel activity of NAC. Both NAC and catalase, another known scavenger of ROS, similarly inhibited ROS levels and apoptosis associated with H₂O₂. However, only NAC, and not catalase or another ROS scavenger Trolox, was able to prevent effects linked to proteasome inhibition, such as protein stabilization, apoptosis and accumulation of ubiquitin conjugates. These observations suggest that NAC has a dual activity as an inhibitor of ROS and proteasome inhibitors. Recently, NAC was used as a ROS inhibitor to functionally characterize a novel anticancer compound, piperlongumine, leading to its description as a ROS inducer. In contrast, our own experiments showed that this compound depicts features of proteasome inhibitors including suppression of FOXM1 (Forkhead box protein M1), stabilization of cellular proteins, induction of ROS-independent apoptosis and enhanced accumulation of ubiquitin conjugates. In addition, NAC, but not catalase or Trolox, interfered with the activity of piperlongumine, further supporting that piperlongumine is a proteasome inhibitor. Most importantly, we showed that NAC, but not other ROS scavengers, directly binds to proteasome inhibitors. To our knowledge, NAC is the first known compound that directly interacts with and antagonizes the activity of proteasome inhibitors. Taken together, the findings of the present study suggest that, as a result of the dual nature of NAC, data interpretation might not be straightforward when NAC is utilized as an antioxidant to demonstrate ROS involvement in drug-induced apoptosis. PMID:23772801

  2. Formation of Tankyrase Inhibitor-Induced Degradasomes Requires Proteasome Activity

    PubMed Central

    Pedersen, Nina Marie; Thorvaldsen, Tor Espen; Schultz, Sebastian Wolfgang; Wenzel, Eva Maria; Stenmark, Harald

    2016-01-01

    In canonical Wnt signaling, the protein levels of the key signaling mediator β-catenin are under tight regulation by the multimeric destruction complex that mediates proteasomal degradation of β-catenin. In colorectal cancer, destruction complex activity is often compromised due to mutations in the multifunctional scaffolding protein Adenomatous Polyposis Coli (APC), leading to a stabilization of β-catenin. Recently, tankyrase inhibitors (TNKSi), a novel class of small molecule inhibitors, were shown to re-establish a functional destruction complex in APC-mutant cancer cell lines by stabilizing AXIN1/2, whose protein levels are usually kept low via poly(ADP-ribosyl)ation by the tankyrase enzymes (TNKS1/2). Surprisingly, we found that for the formation of the morphological correlates of destruction complexes, called degradasomes, functional proteasomes are required. In addition we found that AXIN2 is strongly upregulated after 6 h of TNKS inhibition. The proteasome inhibitor MG132 counteracted TNKSi-induced degradasome formation and AXIN2 stabilization, and this was accompanied by reduced transcription of AXIN2. Mechanistically we could implicate the transcription factor FoxM1 in this process, which was recently shown to be a transcriptional activator of AXIN2. We observed a substantial reduction in TNKSi-induced stabilization of AXIN2 after siRNA-mediated depletion of FoxM1 and found that proteasome inhibition reduced the active (phosphorylated) fraction of FoxM1. This can explain the decreased protein levels of AXIN2 after MG132 treatment. Our findings have implications for the design of in vitro studies on the destruction complex and for clinical applications of TNKSi. PMID:27482906

  3. Associated degeneration of ventral tegmental area dopaminergic neurons in the rat nigrostriatal lactacystin model of parkinsonism and their neuroprotection by valproate

    PubMed Central

    Harrison, Ian F.; Anis, Hiba K.; Dexter, David T.

    2016-01-01

    Parkinson’s disease (PD) manifests clinically as bradykinesia, rigidity, and development of a resting tremor, primarily due to degeneration of dopaminergic nigrostriatal pathways in the brain. Intranigral administration of the irreversible ubiquitin proteasome system inhibitor, lactacystin, has been used extensively to model nigrostriatal degeneration in rats, and study the effects of candidate neuroprotective agents on the integrity of the dopaminergic nigrostriatal system. Recently however, adjacent extra-nigral brain regions such as the ventral tegmental area (VTA) have been noted to also become affected in this model, yet their integrity in studies of candidate neuroprotective agents in the model have largely been overlooked. Here we quantify the extent and distribution of dopaminergic degeneration in the VTA of rats intranigrally lesioned with lactacystin, and quantify the extent of VTA dopaminergic neuroprotection after systemic treatment with an epigenetic therapeutic agent, valproate, shown previously to protect dopaminergic SNpc neurons in this model. We found that unilateral intranigral administration of lactacystin resulted in a 53.81% and 31.72% interhemispheric loss of dopaminergic SNpc and VTA neurons, respectively. Daily systemic treatment of lactacystin lesioned rats with valproate however resulted in dose-dependant neuroprotection of VTA neurons. Our findings demonstrate that not only is the VTA also affected in the intranigral lactacystin rat model of PD, but that this extra-nigral brain region is substrate for neuroprotection by valproate, an agent shown previously to induce neuroprotection and neurorestoration of SNpc dopaminergic neurons in this model. Our results therefore suggest that valproate is a candidate for extra-nigral as well as intra-nigral neuroprotection. PMID:26742637

  4. Associated degeneration of ventral tegmental area dopaminergic neurons in the rat nigrostriatal lactacystin model of parkinsonism and their neuroprotection by valproate.

    PubMed

    Harrison, Ian F; Anis, Hiba K; Dexter, David T

    2016-02-12

    Parkinson's disease (PD) manifests clinically as bradykinesia, rigidity, and development of a resting tremor, primarily due to degeneration of dopaminergic nigrostriatal pathways in the brain. Intranigral administration of the irreversible ubiquitin proteasome system inhibitor, lactacystin, has been used extensively to model nigrostriatal degeneration in rats, and study the effects of candidate neuroprotective agents on the integrity of the dopaminergic nigrostriatal system. Recently however, adjacent extra-nigral brain regions such as the ventral tegmental area (VTA) have been noted to also become affected in this model, yet their integrity in studies of candidate neuroprotective agents in the model have largely been overlooked. Here we quantify the extent and distribution of dopaminergic degeneration in the VTA of rats intranigrally lesioned with lactacystin, and quantify the extent of VTA dopaminergic neuroprotection after systemic treatment with an epigenetic therapeutic agent, valproate, shown previously to protect dopaminergic SNpc neurons in this model. We found that unilateral intranigral administration of lactacystin resulted in a 53.81% and 31.72% interhemispheric loss of dopaminergic SNpc and VTA neurons, respectively. Daily systemic treatment of lactacystin lesioned rats with valproate however resulted in dose-dependant neuroprotection of VTA neurons. Our findings demonstrate that not only is the VTA also affected in the intranigral lactacystin rat model of PD, but that this extra-nigral brain region is substrate for neuroprotection by valproate, an agent shown previously to induce neuroprotection and neurorestoration of SNpc dopaminergic neurons in this model. Our results therefore suggest that valproate is a candidate for extra-nigral as well as intra-nigral neuroprotection. PMID:26742637

  5. Early increase in dopamine release in the ipsilateral striatum after unilateral intranigral administration of lactacystin produces spontaneous contralateral rotations in rats.

    PubMed

    Konieczny, J; Lenda, T; Czarnecka, A

    2016-06-01

    Since the discovery of the role of the ubiquitin-proteasome system (UPS) in the pathogenesis of Parkinson's disease, UPS inhibitors, such as lactacystin have been used to investigate the relationship between UPS impairment and degeneration of dopamine (DA) neurons. However, mostly long-term neurotoxic effects of lactacystin have been studied in animal models. Therefore, the aim of our study was to investigate behavioral and biochemical changes related to the DA system during the first week following unilateral intranigral injection of lactacystin to rats. We found that lactacystin produced early spontaneous contralateral rotations which were inhibited by combined administration of DA D1 and D2 receptor antagonists. Simultaneously, an increase in the extracellular level of DA and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA) was found in the ipsilateral striatum. In contrast, one week after lesion, when turning behavior was no longer visible, a decrease in the extracellular level of DA, DOPAC and HVA was demonstrated. It was accompanied by a substantial reduction in the tissue levels of DA and its metabolites in the lesioned substantia nigra and striatum. We concluded that unilateral intranigral administration of lactacystin produces an early increase in DA neurotransmission which precedes a decrease in the striatal and nigral tissue DA content. It is manifested by the appearance of spontaneous contralateral rotations and an elevation of the extracellular DA level in the ipsilateral striatum. Since similar behavior was previously observed after intranigral administration of rotenone and MPP(+) but not 6-hydroxydopamine (6-OHDA), it may indicate a common mechanism of action shared by these neurotoxins. PMID:26964686

  6. Structure-Driven Developments of 26S Proteasome Inhibitors.

    PubMed

    Śledź, Paweł; Baumeister, Wolfgang

    2016-01-01

    The 26S proteasome is a 2.5-MDa complex, and it operates at the executive end of the ubiquitin-proteasome pathway. It is a proven target for therapeutic agents for the treatment of some cancers and autoimmune diseases, and moreover, it has potential as a target of antibacterial agents. Most inhibitors, including all molecules approved for clinical use, target the 20S proteolytic core complex; its structure was determined two decades ago. Hitherto, efforts to develop inhibitors targeting the 19S regulatory particle subunits have been less successful. This is, in part, because the molecular architecture of this subcomplex has been, until recently, poorly understood, and high-resolution structures have been available only for a few subunits. In this review, we describe, from a structural perspective, the development of inhibitory molecules that target both the 20S and 19S subunits of the proteasome. We highlight the recent progress achieved in structure-based drug-discovery approaches, and we discuss the prospects for further improvement. PMID:26738474

  7. Marizomib, a potent second generation proteasome inhibitor from natural origin.

    PubMed

    Ma, Long; Diao, Aipo

    2015-01-01

    The malignance of cancers reinforces the need to find potent antineoplastic agents. In the past decades, proteasome has been witnessed as a potential target to fulfil this purpose, as evidenced by the fact that the first-in-class proteasome inhibitor Bortezomib was marketed in 2003. Marizomib (Salinosporamide A, NPI-0052), as a marine natural product, promises to be of high efficacy against multiple myeloma (MM), relapsed/refractory MM and other types of solid tumours. Compared with Bortezomib, it arguably has fewer severe side effects. Marizomib has been termed as orphan drug against multiple myeloma by US Food and Drug Administration (FDA) in 2013 and by European Medicines Agency (EMA) in 2014. As one of the second generation proteasome inhibitors (PIs), Marizomib is expected to bring about a sustained and complete therapeutic to extend cancer patients' life span. In this article, we intended to briefly review the historical developments, mechanisms, pharmacology, biosynthesis and side effects of this agent, aiming to provide concise coverage for a broad readership. In the end, we proposed our perspective for its futuristic applications. PMID:25403165

  8. A novel proteasome inhibitor suppresses tumor growth via targeting both 19S proteasome deubiquitinases and 20S proteolytic peptidases

    PubMed Central

    Liu, Ningning; Liu, Chunjiao; Li, Xiaofen; Liao, Siyan; Song, Wenbin; Yang, Changshan; Zhao, Chong; Huang, Hongbiao; Guan, Lixia; Zhang, Peiquan; Liu, Shouting; Hua, Xianliang; Chen, Xin; Zhou, Ping; Lan, Xiaoying; Yi, Songgang; Wang, Shunqing; Wang, Xuejun; Dou, Q. Ping; Liu, Jinbao

    2014-01-01

    The successful development of bortezomib-based therapy for treatment of multiple myeloma has established proteasome inhibition as an effective therapeutic strategy, and both 20S proteasome peptidases and 19S deubiquitinases (DUBs) are becoming attractive targets of cancer therapy. It has been reported that metal complexes, such as copper complexes, inhibit tumor proteasome. However, the involved mechanism of action has not been fully characterized. Here we report that (i) copper pyrithione (CuPT), an alternative to tributyltin for antifouling paint biocides, inhibits the ubiquitin-proteasome system (UPS) via targeting both 19S proteasome-specific DUBs and 20S proteolytic peptidases with a mechanism distinct from that of the FDA-approved proteasome inhibitor bortezomib; (ii) CuPT potently inhibits proteasome-specific UCHL5 and USP14 activities; (iii) CuPT inhibits tumor growth in vivo and induces cytotoxicity in vitro and ex vivo. This study uncovers a novel class of dual inhibitors of DUBs and proteasome and suggests a potential clinical strategy for cancer therapy. PMID:24912524

  9. A novel proteasome inhibitor suppresses tumor growth via targeting both 19S proteasome deubiquitinases and 20S proteolytic peptidases.

    PubMed

    Liu, Ningning; Liu, Chunjiao; Li, Xiaofen; Liao, Siyan; Song, Wenbin; Yang, Changshan; Zhao, Chong; Huang, Hongbiao; Guan, Lixia; Zhang, Peiquan; Liu, Shouting; Hua, Xianliang; Chen, Xin; Zhou, Ping; Lan, Xiaoying; Yi, Songgang; Wang, Shunqing; Wang, Xuejun; Dou, Q Ping; Liu, Jinbao

    2014-01-01

    The successful development of bortezomib-based therapy for treatment of multiple myeloma has established proteasome inhibition as an effective therapeutic strategy, and both 20S proteasome peptidases and 19S deubiquitinases (DUBs) are becoming attractive targets of cancer therapy. It has been reported that metal complexes, such as copper complexes, inhibit tumor proteasome. However, the involved mechanism of action has not been fully characterized. Here we report that (i) copper pyrithione (CuPT), an alternative to tributyltin for antifouling paint biocides, inhibits the ubiquitin-proteasome system (UPS) via targeting both 19S proteasome-specific DUBs and 20S proteolytic peptidases with a mechanism distinct from that of the FDA-approved proteasome inhibitor bortezomib; (ii) CuPT potently inhibits proteasome-specific UCHL5 and USP14 activities; (iii) CuPT inhibits tumor growth in vivo and induces cytotoxicity in vitro and ex vivo. This study uncovers a novel class of dual inhibitors of DUBs and proteasome and suggests a potential clinical strategy for cancer therapy. PMID:24912524

  10. Clinical Use of Proteasome Inhibitors in the Treatment of Multiple Myeloma

    PubMed Central

    Merin, Noah M.; Kelly, Kevin R.

    2014-01-01

    Multiple myeloma (MM) is an incurable hematological malignancy characterized by the clonal proliferation of neoplastic plasma cells. The use of proteasome inhibitors in the treatment of MM has led to significant improvements in outcomes. This article reviews data on the use of the two approved proteasome inhibitors (bortezomib and carlfilzomib), as well as newer agents under development. Emphasis is placed on the clinical use of proteasome inhibitors, including management of side effects and combination with other agents. PMID:25545164

  11. MG132, a proteasome inhibitor, induces apoptosis in tumor cells.

    PubMed

    Guo, Na; Peng, Zhilan

    2013-03-01

    The balance between cell proliferation and apoptosis is critical for normal development and for the maintenance of homeostasis in adult organisms. Disruption of this balance has been implicated in a large number of disease processes, ranging from autoimmunity and neurodegenerative disorders to cancer. The ubiquitin-proteasome pathway, responsible for mediating the majority of intracellular proteolysis, plays a crucial role in the regulation of many normal cellular processes, including the cell cycle, differentiation and apoptosis. Apoptosis in cancer cells is closely connected with the activity of ubiquitin-proteasome pathway. The peptide-aldehyde proteasome inhibitor MG132 (carbobenzoxyl-L-leucyl-L-leucyl-L-leucine) induces the apoptosis of cells by a different intermediary pathway. Although the pathway of induction of apoptosis is different, it plays a crucial role in anti-tumor treatment. There are many cancer-related molecules in which the protein levels present in cells are regulated by a proteasomal pathway; for example, tumor inhibitors (P53, E2A, c-Myc, c-Jun, c-Fos), transcription factors (transcription factor nuclear factor-kappa B, IκBα, HIFI, YYI, ICER), cell cycle proteins (cyclin A and B, P27, P21, IAP1/3), MG132 induces cell apoptosis through formation of reactive oxygen species or the upregulation and downregulation of these factors, which is ultimately dependent upon the activation of the caspase family of cysteine proteases. In this article we review the mechanism of the induction of apoptosis in order to provide information required for research. PMID:22897979

  12. Selective Restriction of Nef-Defective Human Immunodeficiency Virus Type 1 by a Proteasome-Dependent Mechanism▿

    PubMed Central

    Qi, Mingli; Aiken, Christopher

    2007-01-01

    The Nef protein enhances human immunodeficiency virus type 1 (HIV-1) infectivity by facilitating an early postentry step in the virus life cycle. We report here that the addition of MG132 or lactacystin, each a specific inhibitor of cellular proteasome activity, preferentially enhances cellular permissiveness to infection by Nef-defective versus wild-type HIV-1. Pseudotyping by the glycoprotein of vesicular stomatitis virus rendered Nef-defective HIV-1 particles minimally responsive to the enhancing effects of proteasome inhibitors. These results suggest that Nef enhances the infectivity of HIV-1 particles by reducing their susceptibility to proteasomal degradation in target cells. PMID:17108041

  13. The cytotoxicity of γ-secretase inhibitor I to breast cancer cells is mediated by proteasome inhibition, not by γ-secretase inhibition

    PubMed Central

    Han, Jianxun; Ma, Ivy; Hendzel, Michael J; Allalunis-Turner, Joan

    2009-01-01

    Introduction Notch is a family of transmembrane protein receptors whose activation requires proteolytic cleavage by γ-secretase. Since aberrant Notch signaling can induce mammary carcinomas in transgenic mice and high expression levels of Notch receptors and ligands correlates with overall poor clinical outcomes, inhibiting γ-secretase with small molecules may be a promising approach for breast cancer treatment. Consistent with this hypothesis, two recent papers reported that γ-secretase inhibitor I (GSI I), Z-LLNle-CHO, is toxic to breast cancer cells both in vitro and in vivo. In this study, we compared the activity and cytotoxicity of Z-LLNle-CHO to that of two highly specific GSIs, DAPT and L-685,458 and three structurally unrelated proteasome inhibitors, MG132, lactacystin, and bortezomib in order to study the mechanism underlying the cytotoxicity of Z-LLNle-CHO in breast cancer cells. Methods Three estrogen receptor (ER) positive cell lines, MCF-7, BT474, and T47D, and three ER negative cell lines, SKBR3, MDA-MB-231, and MDA-MB-468, were used in this study. Both SKBR3 and BT474 cells also overexpress HER2/neu. Cytotoxicity was measured by using an MTS cell viability/proliferation assay. Inhibition of γ-secretase activity was measured by both immunoblotting and immunofluorescent microscopy in order to detect active Notch1 intracellular domain. Proteasome inhibition was determined by using a cell-based proteasome activity assay kit, by immunoblotting to detect accumulation of polyubiquitylated protein, and by immunofluorescent microscopy to detect redistribution of cellular ubiquitin. Results We found that blocking γ-secretase activity by DAPT and L-685,458 had no effect on the survival and proliferation of a panel of six breast cancer cell lines while Z-LLNle-CHO could cause cell death even at concentrations that inhibited γ-secretase activity less efficiently. Furthermore, we observed that Z-LLNle-CHO could inhibit proteasome activity and the relative

  14. Computational Approaches for the Discovery of Human Proteasome Inhibitors: An Overview.

    PubMed

    Guedes, Romina A; Serra, Patrícia; Salvador, Jorge A R; Guedes, Rita C

    2016-01-01

    Proteasome emerged as an important target in recent pharmacological research due to its pivotal role in degrading proteins in the cytoplasm and nucleus of eukaryotic cells, regulating a wide variety of cellular pathways, including cell growth and proliferation, apoptosis, DNA repair, transcription, immune response, and signaling processes. The last two decades witnessed intensive efforts to discover 20S proteasome inhibitors with significant chemical diversity and efficacy. To date, the US FDA approved to market three proteasome inhibitors: bortezomib, carfilzomib, and ixazomib. However new, safer and more efficient drugs are still required. Computer-aided drug discovery has long being used in drug discovery campaigns targeting the human proteasome. The aim of this review is to illustrate selected in silico methods like homology modeling, molecular docking, pharmacophore modeling, virtual screening, and combined methods that have been used in proteasome inhibitors discovery. Applications of these methods to proteasome inhibitors discovery will also be presented and discussed to raise improvements in this particular field. PMID:27438821

  15. Paradoxical resistance of multiple myeloma to proteasome inhibitors by decreased levels of 19S proteasomal subunits

    PubMed Central

    Acosta-Alvear, Diego; Cho, Min Y; Wild, Thomas; Buchholz, Tonia J; Lerner, Alana G; Simakova, Olga; Hahn, Jamie; Korde, Neha; Landgren, Ola; Maric, Irina; Choudhary, Chunaram; Walter, Peter; Weissman, Jonathan S; Kampmann, Martin

    2015-01-01

    Hallmarks of cancer, including rapid growth and aneuploidy, can result in non-oncogene addiction to the proteostasis network that can be exploited clinically. The defining example is the exquisite sensitivity of multiple myeloma (MM) to 20S proteasome inhibitors, such as carfilzomib. However, MM patients invariably acquire resistance to these drugs. Using a next-generation shRNA platform, we found that proteostasis factors, including chaperones and stress-response regulators, controlled the response to carfilzomib. Paradoxically, 19S proteasome regulator knockdown induced resistance to carfilzomib in MM and non-MM cells. 19S subunit knockdown did not affect the activity of the 20S subunits targeted by carfilzomib nor their inhibition by the drug, suggesting an alternative mechanism, such as the selective accumulation of protective factors. In MM patients, lower 19S levels predicted a diminished response to carfilzomib-based therapies. Together, our findings suggest that an understanding of network rewiring can inform development of new combination therapies to overcome drug resistance. DOI: http://dx.doi.org/10.7554/eLife.08153.001 PMID:26327694

  16. Oxidative stress and proteasome inhibitors in multiple myeloma.

    PubMed

    Lipchick, Brittany C; Fink, Emily E; Nikiforov, Mikhail A

    2016-03-01

    Multiple myeloma is a form of plasma cell neoplasm that accounts for approximately 10% of all hematological malignancies. Recently, several novel drugs have been discovered that almost doubled the overall survival of multiple myeloma patients. One of these drugs, the first-in-class proteasome inhibitor bortezomib (Velcade) has demonstrated remarkable response rates in multiple myeloma patients, and yet, currently this disease remains incurable. The major factor undermining the success of multiple myeloma treatment is a rapidly emerging resistance to the available therapy. Thus, the development of stand-alone or adjuvant anti-myeloma agents becomes of paramount importance. Overproduction of intracellular reactive oxygen species (ROS) often accompanies malignant transformation due to oncogene activation and/or enhanced metabolism in tumor cells. As a result, these cells possess higher levels of ROS and lower levels of antioxidant molecules compared to their normal counterparts. Unbalanced production of ROS leads to oxidative stress which, if left unchecked, could be toxic for the cell. In multiple myeloma cells where high rates of immunoglobulin synthesis is an additional factor contributing to overproduction of ROS, further induction of oxidative stress can be an effective strategy to cope with this disease. Here we will review the available data on the role of oxidative stress in the cytotoxicity of proteasome inhibitors and the use of ROS-inducing compounds as anti-myeloma agents. PMID:26827824

  17. Anchanling reduces pathology in a lactacystin- induced Parkinson's disease model☆

    PubMed Central

    Li, Yinghong; Wu, Zhengzhi; Gao, Xiaowei; Zhu, Qingwei; Jin, Yu; Wu, Anmin; Huang, Andrew C. J.

    2012-01-01

    A rat model of Parkinson's disease was induced by injecting lactacystin stereotaxically into the left mesencephalic ventral tegmental area and substantia nigra pars compacta. After rats were intragastrically perfused with Anchanling, a Chinese medicine, mainly composed of magnolol, for 5 weeks, when compared with Parkinson's disease model rats, tyrosine hydroxylase expression was increased, α-synuclein and ubiquitin expression was decreased, substantia nigra cell apoptosis was reduced, and apomorphine-induced rotational behavior was improved. Results suggested that Anchanling can ameliorate Parkinson's disease pathology possibly by enhancing degradation activity of the ubiquitin-proteasome system. PMID:25767493

  18. Progress of computer-aided drug design (CADD) of proteasome inhibitors.

    PubMed

    Lei, Meng; Liu, Yunde; Zhu, Yongqiang; Liu, Zhenming

    2011-12-01

    The target proteasome has been the focus of drug discovery since the first drug bortezomib was launched in 2003. Many structurally diverse proteasome inhibitors were discovered and even some of them entered the clinical trials. Due to rapid technological progress in chemistry, bioinformatics, structural biology and computer technology, computer-aided drug design (CADD) plays a more and more important role in today's drug discovery. Many CADD technologies were employed in designing various inhibitors of proteasome in the past years. This review gives a global description of the development of computer-aided proteasome inhibitor design by using different commercial or academic software. The binding modes of some structurally novel inhibitors with proteasome were visualized with these new technologies. PMID:21824106

  19. Structure- and function-based design of Plasmodium-selective proteasome inhibitors.

    PubMed

    Li, Hao; O'Donoghue, Anthony J; van der Linden, Wouter A; Xie, Stanley C; Yoo, Euna; Foe, Ian T; Tilley, Leann; Craik, Charles S; da Fonseca, Paula C A; Bogyo, Matthew

    2016-02-11

    The proteasome is a multi-component protease complex responsible for regulating key processes such as the cell cycle and antigen presentation. Compounds that target the proteasome are potentially valuable tools for the treatment of pathogens that depend on proteasome function for survival and replication. In particular, proteasome inhibitors have been shown to be toxic for the malaria parasite Plasmodium falciparum at all stages of its life cycle. Most compounds that have been tested against the parasite also inhibit the mammalian proteasome, resulting in toxicity that precludes their use as therapeutic agents. Therefore, better definition of the substrate specificity and structural properties of the Plasmodium proteasome could enable the development of compounds with sufficient selectivity to allow their use as anti-malarial agents. To accomplish this goal, here we use a substrate profiling method to uncover differences in the specificities of the human and P. falciparum proteasome. We design inhibitors based on amino-acid preferences specific to the parasite proteasome, and find that they preferentially inhibit the β2-subunit. We determine the structure of the P. falciparum 20S proteasome bound to the inhibitor using cryo-electron microscopy and single-particle analysis, to a resolution of 3.6 Å. These data reveal the unusually open P. falciparum β2 active site and provide valuable information about active-site architecture that can be used to further refine inhibitor design. Furthermore, consistent with the recent finding that the proteasome is important for stress pathways associated with resistance of artemisinin family anti-malarials, we observe growth inhibition synergism with low doses of this β2-selective inhibitor in artemisinin-sensitive and -resistant parasites. Finally, we demonstrate that a parasite-selective inhibitor could be used to attenuate parasite growth in vivo without appreciable toxicity to the host. Thus, the Plasmodium proteasome is a

  20. Structure and function based design of Plasmodium-selective proteasome inhibitors

    PubMed Central

    Li, Hao; O'Donoghue, Anthony J.; van der Linden, Wouter A.; Xie, Stanley C.; Yoo, Euna; Foe, Ian T.; Tilley, Leann; Craik, Charles S.; da Fonseca, Paula C. A.; Bogyo, Matthew

    2016-01-01

    The proteasome is a multi-component protease complex responsible for regulating key processes such as the cell cycle and antigen presentation1. Compounds that target the proteasome are potentially valuable tools for the treatment of pathogens that depend on proteasome function for survival and replication. In particular, proteasome inhibitors have been shown to be toxic for the malaria parasite Plasmodium falciparum at all stages of its life cycle2-5. Most compounds that have been tested against the parasite also inhibit the mammalian proteasome resulting in toxicity that precludes their use as therapeutic agents2,6. Therefore, better definition of the substrate specificity and structural properties of the Plasmodium proteasome could enable the development of compounds with sufficient selectivity to allow their use as anti-malarial agents. To accomplish this goal, we used a substrate profiling method to uncover differences in the specificities of the human and P. falciparum proteasome. We designed inhibitors based on amino acid preferences specific to the parasite proteasome, and found that they preferentially inhibit the β 2 subunit. We determined the structure of the P. falciparum 20S proteasome bound to the inhibitor using cryo-electron microscopy (cryo-EM) and single particle analysis, to a resolution of 3.6 Å. These data reveal the unusually open P. falciparum β2 active site and provide valuable information regarding active site architecture that can be used to further refine inhibitor design. Furthermore, consistent with the recent finding that the proteasome is important for stress pathways associated with resistance of artemisinin (ART) family anti-malarials7,8, we observed growth inhibition synergism with low doses of this β 2 selective inhibitor in ART sensitive and resistant parasites. Finally, we demonstrated that a parasite selective inhibitor could be used to attenuate parasite growth in vivo without significant toxicity to the host. Thus, the

  1. ACTIVATION OF PERK KINASE IN NEURAL CELLS BY PROTEASOME INHIBITOR TREATMENT

    PubMed Central

    Zhang, Le; Ebenezer, Philip J; Dasuri, Kalavathi; Bruce-Keller, Annadora J.; Fernandez-Kim, Sun Ok; Liu, Ying; Keller, Jeffrey N.

    2010-01-01

    Inhibition of the proteasome proteolytic pathway occurs as the result of normal aging, as well as in a variety of neurodegenerative conditions, and is believed to promote cellular toxicity in each of these conditions through diverse mechanisms. In the present study we examined whether proteasome inhibition alters the protein kinase (PKR)-like ER kinase (PERK). Our studies demonstrate that proteasome inhibitors induce the transient activation of PERK in both primary rat neurons as well as the N2a neural cell line. Experiments with siRNA to PERK demonstrated that the modulation of PERK was not significant involved in regulating toxicity, ubiquitinated protein levels, or ribosome perturbations in response to proteasome inhibitor treatment. Surprisingly, PERK was observed to be involved in the upregulation of p38 kinase following proteasome inhibitor treatment. Taken together, these data demonstrate the ability of proteasome inhibition to activate PERK and demonstrate evidence for novel cross talk between PERK and the activation of p38 kinase in neural cells following proteasome inhibition. Taken together, these data have implications for understanding the basis by which proteasome inhibition alters neural homeostasis, and the basis by which cell signaling cascades are regulated by proteasome inhibition. PMID:19860852

  2. Search for Inhibitors of the Ubiquitin-Proteasome System from Natural Sources for Cancer Therapy.

    PubMed

    Tsukamoto, Sachiko

    2016-01-01

    Since the approval of the proteasome inhibitor, Velcade(®), by the Food and Drug Administration (FDA) for the treatment of relapsed multiple myeloma, inhibitors of the ubiquitin-proteasome system have been attracting increasing attention as promising drug leads for cancer therapy. While the development of drugs for diseases related to this proteolytic system has mainly been achieved by searching libraries of synthetic small molecules or chemical modifications to drug leads, limited searches have been conducted on natural sources. We have been searching natural sources for inhibitors that target this proteolytic system through in-house screening. Our recent studies on the search for natural inhibitors of the ubiquitin-proteasome system, particularly, inhibitors against the proteasome, E1 enzyme (Uba1), E2 enzyme (Ubc13-Uev1A heterodimer), and E3 enzyme (Hdm2), and also those against deubiquitinating enzyme (USP7), are reviewed here. PMID:26833439

  3. Synthesis and evaluation of derivatives of the proteasome deubiquitinase inhibitor b-AP15.

    PubMed

    Wang, Xin; D'Arcy, Pádraig; Caulfield, Thomas R; Paulus, Aneel; Chitta, Kasyapa; Mohanty, Chitralekha; Gullbo, Joachim; Chanan-Khan, Asher; Linder, Stig

    2015-11-01

    The ubiquitin-proteasome system (UPS) is increasingly recognized as a therapeutic target for the development of anticancer therapies. The success of the 20S proteasome core particle (20S CP) inhibitor bortezomib in the clinical management of multiple myeloma has raised the possibility of identifying other UPS components for therapeutic intervention. We previously identified the small molecule b-AP15 as an inhibitor of 19S proteasome deubiquitinase (DUB) activity. Building upon our previous data, we performed a structure-activity relationship (SAR) study on b-AP15 and identified VLX1570 as an analog with promising properties, including enhanced potency and improved solubility in aqueous solution. In silico modeling was consistent with interaction of VLX1570 with key cysteine residues located at the active sites of the proteasome DUBs USP14 and UCHL5. VLX1570 was found to inhibit proteasome deubiquitinase activity in vitro in a manner consistent with competitive inhibition. Furthermore, using active-site-directed probes, VLX1570 also inhibited proteasome DUB activity in exposed cells. Importantly, VLX1570 did not show inhibitory activity on a panel of recombinant non-proteasome DUBs, on recombinant kinases, or on caspase-3 activity, suggesting that VLX1570 is not an overtly reactive general enzyme inhibitor. Taken together, our data shows the chemical and biological properties of VLX1570 as an optimized proteasome DUB inhibitor. PMID:25854145

  4. Suppression of BRCA1 sensitizes cells to proteasome inhibitors

    PubMed Central

    Gu, Y; Bouwman, P; Greco, D; Saarela, J; Yadav, B; Jonkers, J; Kuznetsov, S G

    2014-01-01

    BRCA1 is a multifunctional protein best known for its role in DNA repair and association with breast and ovarian cancers. To uncover novel biologically significant molecular functions of BRCA1, we tested a panel of 198 approved and experimental drugs to inhibit growth of MDA-MB-231 breast cancer cells depleted for BRCA1 by siRNA. 26S proteasome inhibitors bortezomib and carfilzomib emerged as a new class of selective BRCA1-targeting agents. The effect was confirmed in HeLa and U2OS cancer cell lines using two independent siRNAs, and in mouse embryonic stem (ES) cells with inducible deletion of Brca1. Bortezomib treatment did not cause any increase in nuclear foci containing phosphorylated histone H2AX, and knockdown of BRCA2 did not entail sensitivity to bortezomib, suggesting that the DNA repair function of BRCA1 may not be directly involved. We found that a toxic effect of bortezomib on BRCA1-depleted cells is mostly due to deregulated cell cycle checkpoints mediated by RB1-E2F pathway and 53BP1. Similar to BRCA1, depletion of RB1 also conferred sensitivity to bortezomib, whereas suppression of E2F1 or 53BP1 together with BRCA1 reduced induction of apoptosis after bortezomib treatment. A gene expression microarray study identified additional genes activated by bortezomib treatment only in the context of inactivation of BRCA1 including a critical involvement of the ERN1-mediated unfolded protein response. Our data indicate that BRCA1 has a novel molecular function affecting cell cycle checkpoints in a manner dependent on the 26S proteasome activity. PMID:25522274

  5. Suppression of BRCA1 sensitizes cells to proteasome inhibitors.

    PubMed

    Gu, Y; Bouwman, P; Greco, D; Saarela, J; Yadav, B; Jonkers, J; Kuznetsov, S G

    2014-01-01

    BRCA1 is a multifunctional protein best known for its role in DNA repair and association with breast and ovarian cancers. To uncover novel biologically significant molecular functions of BRCA1, we tested a panel of 198 approved and experimental drugs to inhibit growth of MDA-MB-231 breast cancer cells depleted for BRCA1 by siRNA. 26S proteasome inhibitors bortezomib and carfilzomib emerged as a new class of selective BRCA1-targeting agents. The effect was confirmed in HeLa and U2OS cancer cell lines using two independent siRNAs, and in mouse embryonic stem (ES) cells with inducible deletion of Brca1. Bortezomib treatment did not cause any increase in nuclear foci containing phosphorylated histone H2AX, and knockdown of BRCA2 did not entail sensitivity to bortezomib, suggesting that the DNA repair function of BRCA1 may not be directly involved. We found that a toxic effect of bortezomib on BRCA1-depleted cells is mostly due to deregulated cell cycle checkpoints mediated by RB1-E2F pathway and 53BP1. Similar to BRCA1, depletion of RB1 also conferred sensitivity to bortezomib, whereas suppression of E2F1 or 53BP1 together with BRCA1 reduced induction of apoptosis after bortezomib treatment. A gene expression microarray study identified additional genes activated by bortezomib treatment only in the context of inactivation of BRCA1 including a critical involvement of the ERN1-mediated unfolded protein response. Our data indicate that BRCA1 has a novel molecular function affecting cell cycle checkpoints in a manner dependent on the 26S proteasome activity. PMID:25522274

  6. Fellutamide B is a Potent Inhibitor of the Mycobacterium tuberculosis Proteasome

    SciTech Connect

    Lin, G.; Li, D; Chidawanyika, T; Nathan, C; Li, H

    2010-01-01

    Via high-throughput screening of a natural compound library, we have identified a lipopeptide aldehyde, fellutamide B (1), as the most potent inhibitor of the Mycobacterium tuberculosis (Mtb) proteasome tested to date. Kinetic studies reveal that 1 inhibits both Mtb and human proteasomes in a time-dependent manner under steady-state condition. Remarkably, 1 inhibits the Mtb proteasome in a single-step binding mechanism with K{sub i} = 6.8 nM, whereas it inhibits the human proteasome {beta}5 active site following a two-step mechanism with K{sub i} = 11.5 nM and K*{sub i} = 0.93 nM. Co-crystallization of 1 bound to the Mtb proteasome revealed a structural basis for the tight binding of 1 to the active sites of the Mtb proteasome. The hemiacetal group of 1 in the Mtb proteasome takes the (R)-configuration, whereas in the yeast proteasome it takes the (S)-configuration, indicating that the pre-chiral CHO group of 1 binds to the active site Thr1 in a different orientation. Re-examination of the structure of the yeast proteasome in complex with 1 showed significant conformational changes at the substrate-binding cleft along the active site. These structural differences are consistent with the different kinetic mechanisms of 1 against Mtb and human proteasomes.

  7. Characterization of peptidyl boronic acid inhibitors of mammalian 20 S and 26 S proteasomes and their inhibition of proteasomes in cultured cells.

    PubMed Central

    Gardner, R C; Assinder, S J; Christie, G; Mason, G G; Markwell, R; Wadsworth, H; McLaughlin, M; King, R; Chabot-Fletcher, M C; Breton, J J; Allsop, D; Rivett, A J

    2000-01-01

    Proteasomes are large multisubunit proteinases which have several distinct catalytic sites. In this study a series of di- and tri-peptidyl boronic acids have been tested on the chymotrypsin-like activity of purified mammalian 20 S and 26 S proteasomes assayed with succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin (suc-Leu-Leu-Val-Tyr-AMC) as substrate. The inhibition of 20 S proteasomes is competitive but only slowly reversible. The K(i) values for the best inhibitors were in the range 10-100 nM with suc-Leu-Leu-Val-Tyr-AMC as substrate, but the compounds tested were much less effective on other proteasome activities measured with other substrates. Free boronic acid inhibitors exhibited equivalent potency to their pinacol esters. Both benzoyl (Bz)-Phe-boroLeu and benzyloxycarbonyl (Cbz)-Leu-Leu-boroLeu pinacol ester inhibited 20 S and 26 S proteasomes with non-ideal behaviour, differences in inhibition of the two forms of proteasomes becoming apparent at high inhibitor concentrations (above 3xK(i)). Both of these compounds were also potent inhibitors of 20 S and 26 S proteasomes in cultured cells. However, gel filtration of cell extracts prepared from cells treated with radiolabelled phenacetyl-Leu-Leu-boroLeu showed that only 20 S proteasomes were strongly labelled, demonstrating differences in the characteristics of inhibition of 20 S and 26 S proteasomes. The usefulness of peptidyl boronic acid inhibitors for investigations of proteasome-mediated protein degradation was confirmed by the observation that Bz-Phe-boroLeu and Cbz-Leu-Leu-boroLeu pinacol ester inhibited NFkappaB activation with IC(50) values comparable to their K(i) values for purified proteasomes. The latter result supports the view that the chymotrypsin-like activity of proteasomes assayed with suc-Leu-Leu-Val-Tyr-AMC is a critical one for protein degradation in cells. PMID:10677365

  8. Proteasome inhibitors prevent cell death and prolong survival of mice challenged by Shiga toxin

    PubMed Central

    Hattori, Takayuki; Watanabe-Takahashi, Miho; Ohoka, Nobumichi; Hamabata, Takashi; Furukawa, Koichi; Nishikawa, Kiyotaka; Naito, Mikihiko

    2015-01-01

    Shiga toxin (Stx) causes fatal systemic complications. Stx induces apoptosis, but the mechanism of which is unclear. We report that Stx induced rapid reduction of short-lived anti-apoptotic proteins followed by activation of caspase 9 and the progression of apoptosis. Proteasome inhibitors prevented the reduction of anti-apoptotic proteins, and inhibited caspase activation and apoptosis, suggesting that the reduction of anti-apoptotic proteins is a prerequisite for Stx-induced apoptosis. A clinically approved proteasome inhibitor, bortezomib, prolonged the survival of mice challenged by Stx. These results imply that proteasome inhibition may be a novel approach to prevent the fatal effects of Stx. PMID:26273560

  9. Analysing properties of proteasome inhibitors using kinetic and X-ray crystallographic studies.

    PubMed

    Gallastegui, Nerea; Groll, Michael

    2012-01-01

    The combination of X-ray crystallography and kinetic studies of proteasome:ligand complexes has proven to be an important tool in inhibitor analysis of this crucial protein degradation machinery. Here, we describe in detail the purification protocols, proteolytic activity assays, crystallisation methods, and structure determination for the yeast 20S proteasome (CP) in complex with its inhibitors. The fusion of these advanced techniques offers the opportunity to further optimise drugs which are already tested in different clinical phase studies, as well as to design new promising proteasome lead structures which might be suitable for their application in medicine, plant protection, and antibiotics. PMID:22350899

  10. The effects of proteasome inhibitors on bone remodeling in multiple myeloma.

    PubMed

    Zangari, Maurizio; Suva, Larry J

    2016-05-01

    Bone disease is a characteristic feature of multiple myeloma, a malignant plasma cell dyscrasia. In patients with multiple myeloma, the normal process of bone remodeling is dysregulated by aberrant bone marrow plasma cells, resulting in increased bone resorption, prevention of new bone formation, and consequent bone destruction. The ubiquitin-proteasome system, which is hyperactive in patients with multiple myeloma, controls the catabolism of several proteins that regulate bone remodeling. Clinical studies have reported that treatment with the first-in-class proteasome inhibitor bortezomib reduces bone resorption and increases bone formation and bone mineral density in patients with multiple myeloma. Since the introduction of bortezomib in 2003, several next-generation proteasome inhibitors have also been used clinically, including carfilzomib, oprozomib, ixazomib, and delanzomib. This review summarizes the available preclinical and clinical evidence regarding the effect of proteasome inhibitors on bone remodeling in multiple myeloma. PMID:26947893

  11. Nigral proteasome inhibition in mice leads to motor and non-motor deficits and increased expression of Ser129 phosphorylated α-synuclein

    PubMed Central

    Bentea, Eduard; Van der Perren, Anke; Van Liefferinge, Joeri; El Arfani, Anissa; Albertini, Giulia; Demuyser, Thomas; Merckx, Ellen; Michotte, Yvette; Smolders, Ilse; Baekelandt, Veerle; Massie, Ann

    2015-01-01

    Parkinson's disease is a neurodegenerative disorder characterized by motor and non-motor disturbances. Various pathogenic pathways drive disease progression including oxidative stress, mitochondrial dysfunction, α-synuclein aggregation and impairment of protein degradation systems. Dysfunction of the ubiquitin-proteasome system in the substantia nigra of Parkinson's disease patients is believed to be one of the causes of protein aggregation and cell death associated with this disorder. Lactacystin, a potent inhibitor of the proteasome, was previously delivered to the nigrostriatal pathway of rodents to model nigrostriatal degeneration. Although lactacystin-treated animals develop parkinsonian motor impairment, it is currently unknown whether they also develop non-motor symptoms characteristic of this disorder. In order to further describe the proteasome inhibition model of Parkinson's disease, we characterized the unilateral lactacystin model, performed by stereotaxic injection of the toxin in the substantia nigra of mice. We studied the degree of neurodegeneration and the behavioral phenotype 1 and 3 weeks after lactacystin lesion both in terms of motor impairment, as well as non-motor symptoms. We report that unilateral administration of 3 μg lactacystin to the substantia nigra of mice leads to partial (~40%) dopaminergic cell loss and concurrent striatal dopamine depletion, accompanied by increased expression of Ser129-phosphorylated α-synuclein. Behavioral characterization of the model revealed parkinsonian motor impairment, as well as signs of non-motor disturbances resembling early stage Parkinson's disease including sensitive and somatosensory deficits, anxiety-like behavior, and perseverative behavior. The consistent finding of good face validity, together with relevant construct validity, warrant a further evaluation of proteasome inhibition models of Parkinson's disease in pre-clinical research and validation of therapeutic targets. PMID:25873870

  12. Proteasome inhibitor MG-132 lowers gastric adenocarcinoma TMK1 cell proliferation via bone morphogenetic protein signaling

    SciTech Connect

    Wu, William Ka Kei; Sung, Joseph Jao Yiu; Yu Le; Cho, C.H.

    2008-06-27

    Proteasome inhibitor is a novel class of cancer therapeutics, of which the mechanism of action is not fully understood. It is reported that proteasome inhibitor enhances bone morphogenetic protein (BMP) signaling in osteoblasts to stimulate bone formation. BMP signaling is also an important tumor-suppressing pathway in gastric carcinogenesis. We therefore sought to determine the anti-mitogenic effect of proteasome inhibition in relation to BMP signaling in gastric cancer cells. Results showed that proteasome inhibitor MG-132 significantly suppressed the proliferation and the colony-forming ability of gastric cancer TMK1 cells. In this connection, MG-132 activated BMP signaling, manifested as an increase in Smad1/5/8 phosphorylation and up-regulation of p21{sup Waf1/Cip1} mRNA and protein expression. Knockdown of BMP receptor II by RNA interference abolished Smad1/5/8 phosphorylation, p21{sup Waf1/Cip1} induction, and the inhibition of cell proliferation induced by MG-132. Further analysis revealed that MG-132 up-regulated the expression of BMP1 and BMP4 and suppressed the expression of Smad6. Knockdown of Smad6 also mimicked the effect of MG-132 on BMP signaling. Collectively, these findings suggest that inhibition of proteasome suppresses gastric cancer cell proliferation via activation of BMP signaling. This discovery may open up a novel therapeutic avenue to proteasome inhibitors for the management of gastric cancer.

  13. Induction of autophagy by proteasome inhibitor is associated with proliferative arrest in colon cancer cells

    SciTech Connect

    Wu, William Ka Kei Wu Yachun; Yu Le; Li Zhijie; Sung, Joseph Jao Yiu; Cho, C.H.

    2008-09-19

    The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Blockade of UPS by proteasome inhibitors has been shown to activate autophagy. Recent evidence also suggests that proteasome inhibitors may inhibit cancer growth. In this study, the effect of a proteasome inhibitor MG-132 on the proliferation and autophagy of cultured colon cancer cells (HT-29) was elucidated. Results showed that MG-132 inhibited HT-29 cell proliferation and induced G{sub 2}/M cell cycle arrest which was associated with the formation of LC3{sup +} autophagic vacuoles and the accumulation of acidic vesicular organelles. MG-132 also increased the protein expression of LC3-I and -II in a time-dependent manner. In this connection, 3-methyladenine, a Class III phosphoinositide 3-kinase inhibitor, significantly abolished the formation of LC3{sup +} autophagic vacuoles and the expression of LC3-II but not LC3-I induced by MG-132. Taken together, this study demonstrates that inhibition of proteasome in colon cancer cells lowers cell proliferation and activates autophagy. This discovery may shed a new light on the novel function of proteasome in the regulation of autophagy and proliferation in colon cancer cells.

  14. Regulation of dimethyl-fumarate toxicity by proteasome inhibitors.

    PubMed

    Booth, Laurence; Cruickshanks, Nichola; Tavallai, Seyedmehrad; Roberts, Jane L; Peery, Matthew; Poklepovic, Andrew; Dent, Paul

    2014-01-01

    The present studies examined the biology of the multiple sclerosis drug dimethyl-fumarate (DMF) or its in vivo breakdown product and active metabolite mono-methyl-fumarate (MMF), alone or in combination with proteasome inhibitors, in primary human glioblastoma (GBM) cells. MMF enhanced velcade and carfilzomib toxicity in multiple primary GBM isolates. Similar data were obtained in breast and colon cancer cells. MMF reduced the invasiveness of GBM cells, and enhanced the toxicity of ionizing radiation and temozolomide. MMF killed freshly isolated activated microglia which was associated with reduced IL-6, TGFβ and TNFα production. The combination of MMF and the multiple sclerosis drug Gilenya further reduced both GBM and activated microglia viability and cytokine production. Over-expression of c-FLIP-s or BCL(-)XL protected GBM cells from MMF and velcade toxicity. MMF and velcade increased plasma membrane localization of CD95, and knock down of CD95 or FADD blocked the drug interaction. The drug combination inactivated AKT, ERK1/2 and mTOR. Molecular inhibition of AKT/ERK/mTOR signaling enhanced drug combination toxicity whereas molecular activation of these pathways suppressed killing. MMF and velcade increased the levels of autophagosomes and autolysosomes and knock down of ATG5 or Beclin1 protected cells. Inhibition of the eIF2α/ATF4 arm or the IRE1α/XBP1 arm of the ER stress response enhanced drug combination lethality. This was associated with greater production of reactive oxygen species and quenching of ROS suppressed cell killing. PMID:25482938

  15. Validation of the 2nd Generation Proteasome Inhibitor Oprozomib for Local Therapy of Pulmonary Fibrosis

    PubMed Central

    Semren, Nora; Habel-Ungewitter, Nunja C.; Fernandez, Isis E.; Königshoff, Melanie; Eickelberg, Oliver; Stöger, Tobias; Meiners, Silke

    2015-01-01

    Proteasome inhibition has been shown to prevent development of fibrosis in several organs including the lung. However, effects of proteasome inhibitors on lung fibrosis are controversial and cytotoxic side effects of the overall inhibition of proteasomal protein degradation cannot be excluded. Therefore, we hypothesized that local lung-specific application of a novel, selective proteasome inhibitor, oprozomib (OZ), provides antifibrotic effects without systemic toxicity in a mouse model of lung fibrosis. Oprozomib was first tested on the human alveolar epithelial cancer cell line A549 and in primary mouse alveolar epithelial type II cells regarding its cytotoxic effects on alveolar epithelial cells and compared to the FDA approved proteasome inhibitor bortezomib (BZ). OZ was less toxic than BZ and provided high selectivity for the chymotrypsin-like active site of the proteasome. In primary mouse lung fibroblasts, OZ showed significant anti-fibrotic effects, i.e. reduction of collagen I and α smooth muscle actin expression, in the absence of cytotoxicity. When applied locally into the lungs of healthy mice via instillation, OZ was well tolerated and effectively reduced proteasome activity in the lungs. In bleomycin challenged mice, however, locally applied OZ resulted in accelerated weight loss and increased mortality of treated mice. Further, OZ failed to reduce fibrosis in these mice. While upon systemic application OZ was well tolerated in healthy mice, it rather augmented instead of attenuated fibrotic remodelling of the lung in bleomycin challenged mice. To conclude, low toxicity and antifibrotic effects of OZ in pulmonary fibroblasts could not be confirmed for pulmonary fibrosis of bleomycin-treated mice. In light of these data, the use of proteasome inhibitors as therapeutic agents for the treatment of fibrotic lung diseases should thus be considered with caution. PMID:26340365

  16. Validation of the 2nd Generation Proteasome Inhibitor Oprozomib for Local Therapy of Pulmonary Fibrosis.

    PubMed

    Semren, Nora; Habel-Ungewitter, Nunja C; Fernandez, Isis E; Königshoff, Melanie; Eickelberg, Oliver; Stöger, Tobias; Meiners, Silke

    2015-01-01

    Proteasome inhibition has been shown to prevent development of fibrosis in several organs including the lung. However, effects of proteasome inhibitors on lung fibrosis are controversial and cytotoxic side effects of the overall inhibition of proteasomal protein degradation cannot be excluded. Therefore, we hypothesized that local lung-specific application of a novel, selective proteasome inhibitor, oprozomib (OZ), provides antifibrotic effects without systemic toxicity in a mouse model of lung fibrosis. Oprozomib was first tested on the human alveolar epithelial cancer cell line A549 and in primary mouse alveolar epithelial type II cells regarding its cytotoxic effects on alveolar epithelial cells and compared to the FDA approved proteasome inhibitor bortezomib (BZ). OZ was less toxic than BZ and provided high selectivity for the chymotrypsin-like active site of the proteasome. In primary mouse lung fibroblasts, OZ showed significant anti-fibrotic effects, i.e. reduction of collagen I and α smooth muscle actin expression, in the absence of cytotoxicity. When applied locally into the lungs of healthy mice via instillation, OZ was well tolerated and effectively reduced proteasome activity in the lungs. In bleomycin challenged mice, however, locally applied OZ resulted in accelerated weight loss and increased mortality of treated mice. Further, OZ failed to reduce fibrosis in these mice. While upon systemic application OZ was well tolerated in healthy mice, it rather augmented instead of attenuated fibrotic remodelling of the lung in bleomycin challenged mice. To conclude, low toxicity and antifibrotic effects of OZ in pulmonary fibroblasts could not be confirmed for pulmonary fibrosis of bleomycin-treated mice. In light of these data, the use of proteasome inhibitors as therapeutic agents for the treatment of fibrotic lung diseases should thus be considered with caution. PMID:26340365

  17. Proteasome inhibitors attenuated cholesterol-induced cardiac hypertrophy in H9c2 cells.

    PubMed

    Lee, Hyunjung; Park, Jinyoung; Kim, Eunice EunKyeong; Yoo, Young Sook; Song, Eun Joo

    2016-05-01

    The Ubiquitin proteasome system (UPS) plays roles in protein degradation, cell cycle control, and growth and inflammatory cell signaling. Dysfunction of UPS in cardiac diseases has been seen in many studies. Cholesterol acts as an inducer of cardiac hypertrophy. In this study, the effect of proteasome inhibitors on the cholesterol-induced hypertrophic growth in H9c2 cells is examined in order to observe whether UPS is involved in cardiac hypertrophy. The treatment of proteasome inhibitors MG132 and Bortezomib markedly reduced cellular surface area and mRNA expression of β-MHC in cholesterol-induced cardiac hypertrophy. In addition, activated AKT and ERK were significantly attenuated by MG132 and Bortezomib in cholesterol- induced cardiac hypertrophy. We demonstrated that cholesterol- induced cardiac hypertrophy was suppressed by proteasome inhibitors. Thus, regulatory mechanism of cholesterol- induced cardiac hypertrophy by proteasome inhibitors may provide a new therapeutic strategy to prevent the progression of heart failure. [BMB Reports 2016; 49(5): 270-275]. PMID:26592933

  18. Lactacystin inhibits 3T3-L1 adipocyte differentiation through induction of CHOP-10 expression

    SciTech Connect

    Li Xi; Huang Haiyan |; Chen Jiegen; Jiang Lin; Liu Honglei |; Liu Deguo; Song Tanjing; He Qun; Ma Chungu; Ma Duan |; Song Houyan; Tang Qiqun ||. E-mail: qqtang@shmu.edu.cn

    2006-11-10

    Hormonal induction triggers a cascade leading to the expression of CCAAT/enhancer-binding protein(C/EBP){alpha} and peroxisome proliferator-activated receptor (PPAR) {gamma}, C/EBP{alpha}, and PPAR{gamma} turns on series of adipocyte genes that give rise to the adipocyte phenotype. Previous findings indicate that C/EBP{beta}, a transcriptional activator of the C/EBP{alpha} and PPAR{gamma} genes, is rapidly expressed after induction, but lacks DNA-binding activity and therefore cannot activate transcription of the C/EBP{alpha} and PPAR{gamma} genes early in the differentiation program. Acquisition of DNA-binding activity of C/EBP{beta} occurs when CHOP-10, a dominant-negative form of C/EBP family members, is down-regulated and becomes hyperphosphorylated as preadipocytes traverse the G{sub 1}-S checkpoint of mitotic clonal expansion. Evidences are presented in this report that lactacystin, a proteasome inhibitor, up-regulated the CHOP-10 expression, blocked the DNA-binding activity of C/EBP{beta}, and subsequently inhibited MCE as well as adipocyte differentiation.

  19. Induction of Tumor Cell Apoptosis by a Proteasome Deubiquitinase Inhibitor Is Associated with Oxidative Stress

    PubMed Central

    Brnjic, Slavica; Mazurkiewicz, Magdalena; Fryknäs, Mårten; Sun, Chao; Zhang, Xiaonan; Larsson, Rolf

    2014-01-01

    Abstract Aims: b-AP15 is a recently described inhibitor of the USP14/UCHL5 deubiquitinases (DUBs) of the 19S proteasome. Exposure to b-AP15 results in blocking of proteasome function and accumulation of polyubiquitinated protein substrates in cells. This novel mechanism of proteasome inhibition may potentially be exploited for cancer therapy, in particular for treatment of malignancies resistant to currently used proteasome inhibitors. The aim of the present study was to characterize the cellular response to b-AP15-mediated proteasome DUB inhibition. Results: We report that b-AP15 elicits a similar, but yet distinct, cellular response as the clinically used proteasome inhibitor bortezomib. b-AP15 induces a rapid apoptotic response, associated with enhanced induction of oxidative stress and rapid activation of Jun-N-terminal kinase 1/2 (JNK)/activating protein-1 signaling. Scavenging of reactive oxygen species and pharmacological inhibition of JNK reduced b-AP15-induced apoptosis. We further report that endoplasmic reticulum (ER) stress is induced by b-AP15 and is involved in apoptosis induction. In contrast to bortezomib, ER stress is associated with induction of α-subunit of eukaryotic initiation factor 2 phosphorylation. Innovation: The findings establish that different modes of proteasome inhibition result in distinct cellular responses, a finding of potential therapeutic importance. Conclusion: Our data show that enhanced oxidative stress and ER stress are major determinants of the strong apoptotic response elicited by the 19S DUB inhibitor b-AP15. Antioxid. Redox Signal. 21, 2271–2285. PMID:24011031

  20. Antitumor effects of tyropeptin-boronic acid derivatives: New proteasome inhibitors

    PubMed Central

    Momose, Isao; Abe, Hikaru; Watanabe, Takumi; Ohba, Shun-ichi; Yamazaki, Kanami; Dan, Shingo; Yamori, Takao; Masuda, Tohru; Nomoto, Akio

    2014-01-01

    The proteasome degrades numerous regulatory proteins that are critical for tumor growth. Thus, proteasome inhibitors are promising antitumor agents. New proteasome inhibitors, such as tyropeptins and tyropeptin-boronic acid derivatives, have a potent inhibitory activity. Here we report the antitumor effects of two new tyropeptin-boronic acid derivatives, AS-06 and AS-29. AS-06 and AS-29 significantly suppress the degradation of the proteasome-sensitive fluorescent proteins in HEK293PS cells, and induce the accumulation of ubiquitinated proteins in human multiple myeloma cells. We show that these derivatives also suppress the degradation of the NF-κB inhibitor IκB-α and the nuclear translocation of NF-κB p65 in multiple myeloma cells, resulting in the inhibition of NF-κB activation. Furthermore, we demonstrate that AS-06 and AS-29 induce apoptosis through the caspase-8 and caspase-9 cascades. In a xenograft mouse model, i.v. administration of tyropeptin-boronic acid derivatives inhibits proteasome in tumors and clearly suppresses tumor growth in mice bearing human multiple myeloma. Our results indicate that tyropeptin-boronic acid derivatives could be lead therapeutic agents against human multiple myeloma. PMID:25251038

  1. Repression of protein translation and mTOR signaling by proteasome inhibitor in colon cancer cells

    SciTech Connect

    Wu, William Ka Kei; Volta, Viviana; Cho, Chi Hin; Wu, Ya Chun; Li, Hai Tao; Yu, Le; Li, Zhi Jie; Sung, Joseph Jao Yiu

    2009-09-04

    Protein homeostasis relies on a balance between protein synthesis and protein degradation. The ubiquitin-proteasome system is a major catabolic pathway for protein degradation. In this respect, proteasome inhibition has been used therapeutically for the treatment of cancer. Whether inhibition of protein degradation by proteasome inhibitor can repress protein translation via a negative feedback mechanism, however, is unknown. In this study, proteasome inhibitor MG-132 lowered the proliferation of colon cancer cells HT-29 and SW1116. In this connection, MG-132 reduced the phosphorylation of mammalian target of rapamycin (mTOR) at Ser2448 and Ser2481 and the phosphorylation of its downstream targets 4E-BP1 and p70/p85 S6 kinases. Further analysis revealed that MG-132 inhibited protein translation as evidenced by the reductions of {sup 35}S-methionine incorporation and polysomes/80S ratio. Knockdown of raptor, a structural component of mTOR complex 1, mimicked the anti-proliferative effect of MG-132. To conclude, we demonstrate that the inhibition of protein degradation by proteasome inhibitor represses mTOR signaling and protein translation in colon cancer cells.

  2. Evaluation of the proteasome inhibitor MLN9708 in preclinical models of human cancer.

    PubMed

    Kupperman, Erik; Lee, Edmund C; Cao, Yueying; Bannerman, Bret; Fitzgerald, Michael; Berger, Allison; Yu, Jie; Yang, Yu; Hales, Paul; Bruzzese, Frank; Liu, Jane; Blank, Jonathan; Garcia, Khristofer; Tsu, Christopher; Dick, Larry; Fleming, Paul; Yu, Li; Manfredi, Mark; Rolfe, Mark; Bolen, Joe

    2010-03-01

    The proteasome was validated as an oncology target following the clinical success of VELCADE (bortezomib) for injection for the treatment of multiple myeloma and recurring mantle cell lymphoma. Consequently, several groups are pursuing the development of additional small-molecule proteasome inhibitors for both hematologic and solid tumor indications. Here, we describe MLN9708, a selective, orally bioavailable, second-generation proteasome inhibitor that is in phase I clinical development. MLN9708 has a shorter proteasome dissociation half-life and improved pharmacokinetics, pharmacodynamics, and antitumor activity compared with bortezomib. MLN9708 has a larger blood volume distribution at steady state, and analysis of 20S proteasome inhibition and markers of the unfolded protein response confirmed that MLN9708 has greater pharmacodynamic effects in tissues than bortezomib. MLN9708 showed activity in both solid tumor and hematologic preclinical xenograft models, and we found a correlation between greater pharmacodynamic responses and improved antitumor activity. Moreover, antitumor activity was shown via multiple dosing routes, including oral gavage. Taken together, these data support the clinical development of MLN9708 for both hematologic and solid tumor indications. PMID:20160034

  3. Proteasome Addiction Defined in Ewing Sarcoma Is Effectively Targeted by a Novel Class of 19S Proteasome Inhibitors.

    PubMed

    Shukla, Neerav; Somwar, Romel; Smith, Roger S; Ambati, Sri; Munoz, Stanley; Merchant, Melinda; D'Arcy, Padraig; Wang, Xin; Kobos, Rachel; Antczak, Christophe; Bhinder, Bhavneet; Shum, David; Radu, Constantin; Yang, Guangbin; Taylor, Barry S; Ng, Charlotte K Y; Weigelt, Britta; Khodos, Inna; de Stanchina, Elisa; Reis-Filho, Jorge S; Ouerfelli, Ouathek; Linder, Stig; Djaballah, Hakim; Ladanyi, Marc

    2016-08-01

    Ewing sarcoma is a primitive round cell sarcoma with a peak incidence in adolescence that is driven by a chimeric oncogene created from the fusion of the EWSR1 gene with a member of the ETS family of genes. Patients with metastatic and recurrent disease have dismal outcomes and need better therapeutic options. We screened a library of 309,989 chemical compounds for growth inhibition of Ewing sarcoma cells to provide the basis for the development of novel therapies and to discover vulnerable pathways that might broaden our understanding of the pathobiology of this aggressive sarcoma. This screening campaign identified a class of benzyl-4-piperidone compounds that selectively inhibit the growth of Ewing sarcoma cell lines by inducing apoptosis. These agents disrupt 19S proteasome function through inhibition of the deubiquitinating enzymes USP14 and UCHL5. Functional genomic data from a genome-wide shRNA screen in Ewing sarcoma cells also identified the proteasome as a node of vulnerability in Ewing sarcoma cells, providing orthologous confirmation of the chemical screen findings. Furthermore, shRNA-mediated silencing of USP14 or UCHL5 in Ewing sarcoma cells produced significant growth inhibition. Finally, treatment of a xenograft mouse model of Ewing sarcoma with VLX1570, a benzyl-4-piperidone compound derivative currently in clinical trials for relapsed multiple myeloma, significantly inhibited in vivo tumor growth. Overall, our results offer a preclinical proof of concept for the use of 19S proteasome inhibitors as a novel therapeutic strategy for Ewing sarcoma. Cancer Res; 76(15); 4525-34. ©2016 AACR. PMID:27256563

  4. Marizomib, a Proteasome Inhibitor for All Seasons: Preclinical Profile and a Framework for Clinical Trials

    PubMed Central

    Potts, B.C.; Albitar, M.X.; Anderson, K.C.; Baritaki, S.; Berkers, C.; Bonavida, B.; Chandra, J.; Chauhan, D.; Cusack, J.C.; Fenical, W.; Ghobrial, I.M.; Groll, M.; Jensen, P.R.; Lam, K.S.; Lloyd, G.K.; McBride, W.; McConkey, D.J.; Miller, C.P.; Neuteboom, S.T.C.; Oki, Y.; Ovaa, H.; Pajonk, F.; Richardson, P.G.; Roccaro, A.M.; Sloss, C.M.; Spear, M.A.; Valashi, E.; Younes, A.; Palladino, M.A.

    2013-01-01

    The proteasome has emerged as an important clinically relevant target for the treatment of hematologic malignancies. Since the Food and Drug Administration approved the first-in-class proteasome inhibitor bortezomib (Velcade®) for the treatment of relapsed/refractory multiple myeloma (MM) and mantle cell lymphoma, it has become clear that new inhibitors are needed that have a better therapeutic ratio, can overcome inherent and acquired bortezomib resistance and exhibit broader anti-cancer activities. Marizomib (NPI-0052; salinosporamide A) is a structurally and pharmacologically unique β-lactone-γ-lactam proteasome inhibitor that may fulfill these unmet needs. The potent and sustained inhibition of all three proteolytic activities of the proteasome by marizomib has inspired extensive preclinical evaluation in a variety of hematologic and solid tumor models, where it is efficacious as a single agent and in combination with biologics, che-motherapeutics and targeted therapeutic agents. Specifically, marizomib has been evaluated in models for multiple myeloma, mantle cell lymphoma, Waldenstrom’s macroglobulinemia, chronic and acute lymphocytic leukemia, as well as glioma, colorectal and pancreatic cancer models, and has exhibited synergistic activities in tumor models in combination with bortezomib, the immunomodulatory agent lenalidomide (Revlimid®), and various histone deacetylase inhibitors. These and other studies provided the framework for ongoing clinical trials in patients with MM, lymphomas, leukemias and solid tumors, including those who have failed bortezomib treatment, as well as in patients with diagnoses where other proteasome inhibitors have not demonstrated significant efficacy. This review captures the remarkable translational studies and contributions from many collaborators that have advanced marizomib from seabed to bench to bedside. PMID:21247382

  5. Antiproliferative and proapoptotic effects of proteasome inhibitors and their combination with histone deacetylase inhibitors on leukemia cells.

    PubMed

    Fuchs, Ota; Provaznikova, Dana; Marinov, Iuri; Kuzelova, Katerina; Spicka, Ivan

    2009-03-01

    New chemotherapeutic agents are still required to further optimise treatment of leukemia patients. Proteasome inhibition by bortezomib, PR-171 (carfilzomib) and NPI-0052 (salinosporamide A) has been successfully used for the treatment of multiple myeloma and mantle cell lymphoma and is considered also as novel treatment strategy in leukemia. Combination of proteasome inhibitors bortezomib and NPI-0052 induces synergistic anti-multiple myeloma activity both in vitro using multiple myeloma cells and in vivo in a human plasmacytoma xenograft mouse model. Cell death resulting from proteasome inhibition requires caspase activation and increased levels of reactive oxygen species. While bortezomib induces several caspases, NPI-0052 activates predominantly caspase-8-dependent pathway. We studied the effect of bortezomib (10 nM) on DNA synthesis and apoptosis in human acute myeloid cell lines KASUMI-1, ML-1, ML-2 and CTV-1 cells. Bortezomib was potent inhibitor of DNA synthesis in all four types of leukemia cells and induced apoptosis in KASUMI-1, ML-2 and CTV-1 cells but not in ML-1 cells. Other research groups showed that histone deacetylase inhibitors (valproic acid or benzamide derivative MS-275) in combination with NPI-0052 or PR-171 induced greater levels of acute leukemia cell death than in combination with bortezomib. Proteasome inhibition as monotherapy and its combination with many conventional therapies as novel treatment strategies in leukemia are promising. Malignant cells are more sensitive to this treatment than normal hematopoietic cells. PMID:19275578

  6. Dithiocarbamate-based coordination compounds as potent proteasome inhibitors in human cancer cells.

    PubMed

    Buac, Daniela; Schmitt, Sara; Ventro, George; Kona, Fathima Rani; Dou, Q Ping

    2012-10-01

    Dithiocarbamates are a class of metal-chelating compounds with various applications in medicine. They have been used for the treatment of bacterial and fungal infections, possible treatment of AIDS, and most recently cancer. Their anti-tumor effects can in part be attributed to their ability to complex tumor cellular copper, leading to binding to and inhibition of the proteasome and in turn initiating tumor cell-specific apoptosis. Current chemotherapeutic agents are highly toxic and therefore their efficacy in the eradication of tumors is greatly limited. As a result many scientists have joined the quest for novel targeted therapies in hopes of reducing toxicity while maximizing potency and proteasome inhibition has become an attractive therapy in this regard. Here we discuss the origins, mechanism, and evolution of dithiocarbamates as potent proteasome inhibitors and therefore anti-cancer agents. PMID:22931591

  7. Dithiocarbamate-Based Coordination Compounds as Potent Proteasome Inhibitors in Human Cancer Cells

    PubMed Central

    Buac, Daniela; Schmitt, Sara; Ventro, George; Kona, Fathima Rani; Dou, Q. Ping

    2013-01-01

    Dithiocarbamates are a class of metal-chelating compounds with various applications in medicine. They have been used for the treatment of bacterial and fungal infections, possible treatment of AIDS, and most recently cancer. Their anti-tumor effects can in part be attributed to their ability to complex tumor cellular copper, leading to binding to and inhibition of the proteasome and in turn initiating tumor cell-specific apoptosis. Current chemotherapeutic agents are highly toxic and therefore their efficacy in the eradication of tumors is greatly limited. As a result many scientists have joined the quest for novel targeted therapies in hopes of reducing toxicity while maximizing potency and proteasome inhibition has become an attractive therapy in this regard. Here we discuss the origins, mechanism, and evolution of dithiocarbamates as potent proteasome inhibitors and therefore anti-cancer agents. PMID:22931591

  8. Proteasome inhibitors – molecular basis and current perspectives in multiple myeloma

    PubMed Central

    Kubiczkova, Lenka; Pour, Ludek; Sedlarikova, Lenka; Hajek, Roman; Sevcikova, Sabina

    2014-01-01

    Inhibition of proteasome, a proteolytic complex responsible for the degradation of ubiquitinated proteins, has emerged as a powerful strategy for treatment of multiple myeloma (MM), a plasma cell malignancy. First-in-class agent, bortezomib, has demonstrated great positive therapeutic efficacy in MM, both in pre-clinical and in clinical studies. However, despite its high efficiency, a large proportion of patients do not achieve sufficient clinical response. Therefore, the development of a second-generation of proteasome inhibitors (PIs) with improved pharmacological properties was needed. Recently, several of these new agents have been introduced into clinics including carfilzomib, marizomib and ixazomib. Further, new orally administered second-generation PI oprozomib is being investigated. This review provides an overview of main mechanisms of action of PIs in MM, focusing on the ongoing development and progress of novel anti-proteasome therapeutics. PMID:24712303

  9. Synthesis and Biological Evaluation of Naphthoquinone Analogs as a Novel Class of Proteasome Inhibitors

    PubMed Central

    Lawrence, Harshani R.; Kazi, Aslamuzzaman; Luo, Yunting; Kendig, Robert; Ge, Yiyu; Jain, Sanjula; Daniel, Kenyon; Santiago, Daniel; Guida, Wayne C.; Sebti, Saïd M.

    2012-01-01

    Screening of the NCI Diversity Set-1 identified PI-083 (NSC-45382) a proteasome inhibitor selective for cancer over normal cells. Focused libraries of novel compounds based on PI-083 chloronaphthoquinone and sulfonamide moieties were synthesized to gain a better understanding of the structure activity relationship responsible for chymotrypsin-like proteasome inhibitory activity. This led to the demonstration that the chloronaphthoquinone and the sulfonamide moieties are critical for inhibitory activity. The pyridyl group in PI-083 can be replaced with other heterocyclic groups without significant loss of activity. Molecular modeling studies were also performed to explore the detailed interactions of PI-083 and its derivatives with the β5 and β6 subunits of the 20S proteasome. The refined model showed an H-bond interaction between the Asp-114 and the sulfonamide moiety of the PI-083 in the β6 subunit. PMID:20621484

  10. Multiple proteolytic systems, including the proteasome, contribute to CFTR processing.

    PubMed

    Jensen, T J; Loo, M A; Pind, S; Williams, D B; Goldberg, A L; Riordan, J R

    1995-10-01

    The molecular components of the quality control system that rapidly degrades abnormal membrane and secretory proteins have not been identified. The cystic fibrosis transmembrane conductance regulator (CFTR) is an integral membrane protein to which this quality control is stringently applied; approximately 75% of the wild-type precursor and 100% of the delta F508 CFTR variant found in most CF patients are rapidly degraded before exiting from the ER. We now show that this ER degradation is sensitive to inhibitors of the cytosolic proteasome, including lactacystin and certain peptide aldehydes. One of the latter compounds, MG-132, also completely blocks the ATP-dependent conversion of the wild-type precursor to the native folded form that enables escape from degradation. Hence, CFTR and presumably other intrinsic membrane proteins are substrates for proteasomal degradation during their maturation within the ER. PMID:7553864

  11. Proteasome dysfunction inhibits surfactant protein gene expression in lung epithelial cells: mechanism of inhibition of SP-B gene expression.

    PubMed

    Das, Aparajita; Boggaram, Vijayakumar

    2007-01-01

    Surfactant proteins maintain lung function through their actions to reduce alveolar surface tension and control of innate immune responses in the lung. The ubiquitin proteasome pathway is responsible for the degradation of majority of intracellular proteins in eukaryotic cells, and proteasome dysfunction has been linked to the development of neurodegenerative, cardiac, and other diseases. Proteasome function is impaired in interstitial lung diseases associated with surfactant protein C (SP-C) mutation mapping to the BRICHOS domain located in the proSP-C protein. In this study we determined the effects of proteasome inhibition on surfactant protein expression in H441 and MLE-12 lung epithelial cells to understand the relationship between proteasome dysfunction and surfactant protein gene expression. Proteasome inhibitors lactacystin and MG132 reduced the levels of SP-A, SP-B, and SP-C mRNAs in a concentration-dependent manner in H441 and MLE-12 cells. In H441 cells, lactacystin and MG132 inhibition of SP-B mRNA was associated with similar decreases in SP-B protein, and the inhibition was due to inhibition of gene transcription. Proteasome inhibitors decreased thyroid transcription factor-1 (TTF-1)/Nkx2.1 DNA binding activity, and the reduced TTF-1 DNA binding activity was due to reduced expression levels of TTF-1 protein. These data indicated that the ubiquitin proteasome pathway is essential for the maintenance of surfactant protein gene expression and that disruption of this pathway inhibits surfactant protein gene expression via reduced expression of TTF-1 protein. PMID:16905641

  12. Inhibition of the proteasome induces cell cycle arrest and apoptosis in mantle cell lymphoma cells.

    PubMed

    Bogner, Christian; Ringshausen, Ingo; Schneller, Folker; Fend, Falko; Quintanilla-Martinez, Leticia; Häcker, Georg; Goetze, Katharina; Oostendorp, Robert; Peschel, Christian; Decker, Thomas

    2003-07-01

    Mantle cell lymphoma (MCL) is a distinctive non-Hodgkin's lymphoma subtype, characterized by overexpression of cyclin D1 as a consequence of the chromosomal translocation t(11;14)(q13;q32). MCL remains an incurable disease, combining the unfavourable clinical features of aggressive and indolent lymphomas. The blastic variant of MCL, which is often associated with additional cytogenetic alterations, has an even worse prognosis and new treatment options are clearly needed. The present study investigated the effect of a specific proteasome inhibitor, lactacystin, on cell cycle progression and apoptosis in two lymphoma cell lines harbouring the t(11;14)(q13;q32) and additional cytogenetic alterations, including p53 mutation (NCEB) and p16 deletion (Granta 519). Granta cells were more susceptible to inhibition of the proteasome with respect to inhibition of proliferation and apoptosis induction. No changes were observed in the expression levels of the G1 regulatory molecules cyclin D1 and cdk4, but cell cycle arrest and apoptosis induction was accompanied by accumulation of the cdk inhibitor p21 in both cell lines. Increased p53 expression was only observed in Granta cells with wild-type p53. Cleavage of procaspase-3 and -9 was observed but cleavage of procaspase-8 was not involved in apoptosis induction. The proapoptotic effect of lactacystin was reversed by pretreatment with the pancaspase inhibitor zVAD.fmk. Lactacystin was also effective in inducing apoptosis in lymphoma cells from MCL patients. We conclude that inhibition of the proteasome might be a promising therapeutic approach for this incurable disease. PMID:12846895

  13. Proteasome inhibitors prevent cytochrome c release during apoptosis but not in excitotoxic death of cerebellar granule neurons.

    PubMed

    Bobba, Antonella; Canu, Nadia; Atlante, Anna; Petragallo, Vito; Calissano, Pietro; Marra, Ersilia

    2002-03-27

    In order to find out whether and how proteasomes participate in the processes leading cerebellar granule cells to death either in necrosis, due to glutamate neurotoxicity, or in apoptosis, due to K(+) shift, we measured the three proteasome activities by using specific fluorescent probes and investigated the effect of several proteasome inhibitors, including MG132, on the cytochrome c release taking place in the early phase of both apoptosis and necrosis. We show that differently from apoptosis, the early phase of necrosis does not require proteasome activation. Inhibition of proteasome activity can prevent cytochrome c release in cerebellar granule cells undergoing apoptosis, thus improving cell survival, but not necrosis. These findings show that proteasomes play an important role in the early phase of apoptosis but not that of necrosis, and that these two types of cell death differ from each other in their mechanism of cytochrome c release. PMID:11943185

  14. Optimization and Evaluation of 5-Styryl-Oxathiazol-2-one Mycobacterium tuberculosis Proteasome Inhibitors as Potential Antitubercular Agents

    PubMed Central

    Russo, Francesco; Gising, Johan; Åkerbladh, Linda; Roos, Annette K; Naworyta, Agata; Mowbray, Sherry L; Sokolowski, Anders; Henderson, Ian; Alling, Torey; Bailey, Mai A; Files, Megan; Parish, Tanya; Karlén, Anders; Larhed, Mats

    2015-01-01

    This is the first report of 5-styryl-oxathiazol-2-ones as inhibitors of the Mycobacterium tuberculosis (Mtb) proteasome. As part of the study, the structure–activity relationship of oxathiazolones as Mtb proteasome inhibitors has been investigated. Furthermore, the prepared compounds displayed a good selectivity profile for Mtb compared to the human proteasome. The 5-styryl-oxathiazol-2-one inhibitors identified showed little activity against replicating Mtb, but were rapidly bactericidal against nonreplicating bacteria. (E)-5-(4-Chlorostyryl)-1,3,4-oxathiazol-2-one) was most effective, reducing the colony-forming units (CFU)/mL below the detection limit in only seven days at all concentrations tested. The results suggest that this new class of Mtb proteasome inhibitors has the potential to be further developed into novel antitubercular agents for synergistic combination therapies with existing drugs. PMID:26246997

  15. The investigational proteasome inhibitor ixazomib for the treatment of multiple myeloma.

    PubMed

    Richardson, Paul G; Moreau, Philippe; Laubach, Jacob P; Gupta, Neeraj; Hui, Ai-Min; Anderson, Kenneth C; San Miguel, Jesús F; Kumar, Shaji

    2015-01-01

    Ixazomib is an investigational, reversible 20S proteasome inhibitor. It is the first oral proteasome inhibitor under clinical investigation in multiple myeloma (MM). Under physiological conditions, the stable citrate ester drug substance, ixazomib citrate (MLN9708), rapidly hydrolyzes to the biologically active boronic acid, ixazomib (MLN2238). Preclinical studies have demonstrated antitumor activity in MM cell lines and xenograft models. In Phase I/II clinical studies ixazomib has had generally manageable toxicities, with limited peripheral neuropathy observed to date. Preliminary data from these studies indicate ixazomib is active as a single agent in relapsed/refractory MM and as part of combination regimens in newly diagnosed patients. Phase III studies in combination with lenalidomide-dexamethasone are ongoing. PMID:25832873

  16. Augmentation of fear extinction by D-cycloserine is blocked by proteasome inhibitors.

    PubMed

    Mao, Sheng-Chun; Lin, Hui-Ching; Gean, Po-Wu

    2008-12-01

    D-Cycloserine (DCS) has been shown to facilitate extinction of conditioned fear in rats and to improve fear reduction of social phobia and fear of heights in human studies. Here, we investigate the mechanism of DCS effect by measuring internalized GluR1 and GluR2 using cell-surface biotinylation techniques. DCS selectively increased NMDA receptor-mediated synaptic response without affecting AMPA receptor-mediated synaptic response. Low-frequency stimulation (LFS) when applied in the presence of DCS induced GluR1 and GluR2 internalization in the amygdala slices. Proteasome inhibitors block DCS facilitation of LFS-induced depotentiation and a reduction in surface levels of GluR1 and GluR2. Furthermore, DCS in combination with LFS reduced cellular levels of PSD-95 and synapse-associated protein 97 (SAP97), which were also blocked by proteasome inhibitors. In the in vivo experiments, DCS-induced reduction of fear-potentiated startle and reversal of conditioning-induced increase in surface expression of GluR1 were blocked by proteasome inhibitors. DCS-treated rats fail to exhibit reinstatement after US-alone presentations. These results suggest that DCS facilitates receptor internalization in the presence of extinction training, resulting in augmented reduction of startle potentiation. PMID:18368037

  17. Discovery of new [Formula: see text] proteasome inhibitors using a knowledge-based computational screening approach.

    PubMed

    Mehra, Rukmankesh; Chib, Reena; Munagala, Gurunadham; Yempalla, Kushalava Reddy; Khan, Inshad Ali; Singh, Parvinder Pal; Khan, Farrah Gul; Nargotra, Amit

    2015-11-01

    Mycobacterium tuberculosis bacteria cause deadly infections in patients [Corrected]. The rise of multidrug resistance associated with tuberculosis further makes the situation worse in treating the disease. M. tuberculosis proteasome is necessary for the pathogenesis of the bacterium validated as an anti-tubercular target, thus making it an attractive enzyme for designing Mtb inhibitors. In this study, a computational screening approach was applied to identify new proteasome inhibitor candidates from a library of 50,000 compounds. This chemical library was procured from the ChemBridge (20,000 compounds) and the ChemDiv (30,000 compounds) databases. After a detailed analysis of the computational screening results, 50 in silico hits were retrieved and tested in vitro finding 15 compounds with [Formula: see text] values ranging from 35.32 to 64.15 [Formula: see text]M on lysate. A structural analysis of these hits revealed that 14 of these compounds probably have non-covalent mode of binding to the target and have not reported for anti-tubercular or anti-proteasome activity. The binding interactions of all the 14 protein-inhibitor complexes were analyzed using molecular docking studies. Further, molecular dynamics simulations of the protein in complex with the two most promising hits were carried out so as to identify the key interactions and validate the structural stability. PMID:26232029

  18. Proteasome inhibitors, including curcumin, improve pancreatic β-cell function and insulin sensitivity in diabetic mice

    PubMed Central

    Weisberg, S; Leibel, R; Tortoriello, D V

    2016-01-01

    Background: Type 2 diabetes stems from obesity-associated insulin resistance, and in the genetically susceptible, concomitant pancreatic β-cell failure can occur, which further exacerbates hyperglycemia. Recent work by our group and others has shown that the natural polyphenol curcumin attenuates the development of insulin resistance and hyperglycemia in mouse models of hyperinsulinemic or compensated type 2 diabetes. Although several potential downstream molecular targets of curcumin exist, it is now recognized to be a direct inhibitor of proteasome activity. We now show that curcumin also prevents β-cell failure in a mouse model of uncompensated obesity-related insulin resistance (Leprdb/db on the Kaliss background). Results: In this instance, dietary supplementation with curcumin prevented hyperglycemia, increased insulin production and lean body mass, and prolonged lifespan. In addition, we show that short-term in vivo treatment with low dosages of two molecularly distinct proteasome inhibitors celastrol and epoxomicin reverse hyperglycemia in mice with β-cell failure by increasing insulin production and insulin sensitivity. Conclusions: These studies suggest that proteasome inhibitors may prove useful for patients with diabetes by improving both β-cell function and relieving insulin resistance. PMID:27110686

  19. Production of Proteasome Inhibitor Syringolin A by the Endophyte Rhizobium sp. Strain AP16

    PubMed Central

    Bigler, Laurent; Dudler, Robert

    2014-01-01

    Syringolin A, the product of a mixed nonribosomal peptide synthetase/polyketide synthase encoded by the syl gene cluster, is a virulence factor secreted by certain Pseudomonas syringae strains. Together with the glidobactins produced by a number of beta- and gammaproteobacterial human and animal pathogens, it belongs to the syrbactins, a structurally novel class of proteasome inhibitors. In plants, proteasome inhibition by syringolin A-producing P. syringae strains leads to the suppression of host defense pathways requiring proteasome activity, such as the ones mediated by salicylic acid and jasmonic acid. Here we report the discovery of a syl-like gene cluster with some unusual features in the alphaproteobacterial endophyte Rhizobium sp. strain AP16 that encodes a putative syringolin A-like synthetase whose components share 55% to 65% sequence identity (72% to 79% similarity) at the amino acid level. As revealed by average nucleotide identity (ANI) calculations, this strain likely belongs to the same species as biocontrol strain R. rhizogenes K84 (formely known as Agrobacterium radiobacter K84), which, however, carries a nonfunctional deletion remnant of the syl-like gene cluster. Here we present a functional analysis of the syl-like gene cluster of Rhizobium sp. strain AP16 and demonstrate that this endophyte synthesizes syringolin A and some related minor variants, suggesting that proteasome inhibition by syrbactin production can be important not only for pathogens but also for endophytic bacteria in the interaction with their hosts. PMID:24727275

  20. Commentary on "Proteasome Inhibitors: A Novel Class of Potent and Effective Antitumor Agents".

    PubMed

    Tew, Kenneth D

    2016-09-01

    The relatively recent clinical success of bortezomib, particularly in multiple myeloma, has established the validity of the proteasome as a viable target for anticancer drug development. This highly cited 1999 Cancer Research article from Adams and colleagues was published during the period when this drug was transitioning from preclinical studies to phase I clinical trial status. Their results detail structure-activity analyses using a series of boronic acid proteasome inhibitors and correlate cytotoxicity with inhibition of proteasome activity. In and of itself, the recognition that interference with proteasome functions represented a novel therapeutic approach likely underlies the popularity of this article. In addition, the provision of in vitro (at that time using the NCI 60 cell line panel) and in vivo antitumor activity, toxicology, and mouse pharmacokinetic and pharmacodynamic data provided a solid basis for establishing the future credentials for bortezomib to gain initial FDA approval in 2003. Cancer Res; 76(17); 4916-7. ©2016 AACRSee related article by Adams et al., Cancer Res 1999;59:2615-22Visit the Cancer Research 75(th) Anniversary timeline. PMID:27587650

  1. Plasminogen Activator Inhibitor Type 1 Interacts with α3 Subunit of Proteasome and Modulates Its Activity*

    PubMed Central

    Boncela, Joanna; Przygodzka, Patrycja; Papiewska-Pajak, Izabela; Wyroba, Elzbieta; Osinska, Magdalena; Cierniewski, Czeslaw S.

    2011-01-01

    Plasminogen activator inhibitor type-1 (PAI-1), a multifunctional protein, is an important physiological regulator of fibrinolysis, extracellular matrix homeostasis, and cell motility. Recent observations show that PAI-1 may also be implicated in maintaining integrity of cells, especially with respect to cellular proliferation or apoptosis. In the present study we provide evidence that PAI-1 interacts with proteasome and affects its activity. First, by using the yeast two-hybrid system, we found that the α3 subunit of proteasome directly interacts with PAI-1. Then, to ensure that the PAI-1-proteasome complex is formed in vivo, both proteins were coimmunoprecipitated from endothelial cells and identified with specific antibodies. The specificity of this interaction was evidenced after transfection of HeLa cells with pCMV-PAI-1 and coimmunoprecipitation of both proteins with anti-PAI-1 antibodies. Subsequently, cellular distribution of the PAI-1-proteasome complexes was established by immunogold staining and electron microscopy analyses. Both proteins appeared in a diffuse cytosolic pattern but also could be found in a dense perinuclear and nuclear location. Furthermore, PAI-1 induced formation of aggresomes freely located in endothelial cytoplasm. Increased PAI-1 expression abrogated degradation of degron analyzed after cotransfection of HeLa cells with pCMV-PAI-1 and pd2EGFP-N1 and prevented degradation of p53 as well as IκBα, as evidenced both by confocal microscopy and Western immunoblotting. PMID:21135093

  2. Proteasome inhibitors exacerbate interleukin-8 production induced by protease-activated receptor 2 in intestinal epithelial cells.

    PubMed

    Ghouzali, Ibtissem; Azhar, Saïda; Bôle-Feysot, Christine; Ducrotté, Philippe; Déchelotte, Pierre; Coëffier, Moïse

    2016-10-01

    Protease activated receptors (PARs) and the ubiquitin-proteasome system (UPS) regulate inflammatory response in intestinal cells. We aimed to elucidate putative connections between PARs and UPS pathways in intestinal epithelial cells. Caco-2 cells were treated by agonist peptides of PARs and/or IL-1β and/or proteasome inhibitors, bortezomib or MG132. Inflammatory response was evaluated by measuring IL-8 production. Proteasome activities were also evaluated. We showed that PAR-1 and -2 activation increased release of IL-8 compared with vehicle and independently of IL-1β. In contrast, PAR-4 agonist peptide had no effect. Caspase-like and chymotrypsin-like proteasomal activities were increased by PAR-2 activation only in the presence of IL-1β. Interestingly, in polarized Caco-2 cells, the release of IL-8 was predominantly upregulated in the side where PAR-2 agonist peptide was added, apical or basalolateral. In contrast, proteasome activities were only affected when PAR-2 agonist peptide was added in the apical side. Proteasome inhibitors, bortezomib and MG132, enhanced IL-8 production in both sides, apical and basolateral. In conclusion, PAR-2 activation alone did not affect proteasome but needed inflammatory stimulus IL-1β to synergistically increase chymotrypsin-like activity in intestinal epithelial cells. However, proteasome inhibition led to exacerbate inflammatory response induced by PAR-2 activation. PMID:27455449

  3. Proteasome inhibitors induce peroxisome proliferator-activated receptor transactivation through RXR accumulation and a protein kinase C-dependent pathway

    SciTech Connect

    Tsao, W.-C.; Wu, H.-M.; Chi, K.-H.; Chang, Y.-H.; Lin, W.-W. . E-mail: wwl@ha.mc.ntu.edu.tw

    2005-03-10

    Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), a member of nuclear hormone receptors, forms a heterodimeric DNA binding complex with retinoid X receptor (RXR) and serves as a transcriptional regulator of gene expression. In this study, using luciferase assay of a reporter gene containing PPAR response element (PPRE), we found PPRE transactivity was additively induced by PPAR{gamma} activator (15dPGJ{sub 2}) and RXR activator (9-cis retinoic acid, 9-cis RA). Proteasome inhibitors MG132 and MG262 also stimulate PPRE transactivity in a concentration-dependent manner, and this effect is synergistic to 15dPGJ{sub 2} and 9-cis RA. PKC activation by 12-myristate 13-acetate (PMA) and ingenol 3,20-dibenzoate (IDB) also led to an increased PPRE activation, and this action was additive to PPAR{gamma} activators and 9-cis RA, but not to proteasome inhibitors. Results indicate that the PPAR{gamma} enhancing effect of proteasome inhibitors was attributed to redox-sensitive PKC activation. Western blot analysis showed that the protein level of RXR{alpha}, but not PPAR{gamma}, RXR{beta}, or PKC isoforms, was accumulated in the presence of proteasome inhibitors. Taken together, we conclude that proteasome inhibitors can upregulate PPRE activity through RXR{alpha} accumulation and a PKC-dependent pathway. The former is due to inhibition of RXR{alpha} degradation through ubiquitin-dependent proteasome system, while the latter is mediated by reactive oxygen species (ROS) production.

  4. Next-generation proteasome inhibitor MLN9708 sensitizes breast cancer cells to doxorubicin-induced apoptosis.

    PubMed

    Wang, Hao; Yu, Yang; Jiang, Zheng; Cao, Wen-Ming; Wang, Zhenyu; Dou, Jun; Zhao, Yanling; Cui, Yunfu; Zhang, Hong

    2016-01-01

    Doxorubicin (Dox), one of the most effective chemotherapy drug for cancer treatment, is limited by its severe side effects and chemoresistance. Dox induces DNA damage and leads to significant proteomic changes in the cancer cells, which makes the ubiquitin-proteasome system a potential target to enhance the efficacy of Dox therapy. The unsuccessful clinical trials of proteasome inhibitor PS-341 (bortezomib) in solid tumors led to the invention of MLN9708 (ixazomib), an orally bioavailable next-generation proteasome inhibitor with improved pharmacokinetic and pharmacodynamic features. In this preclinical study, we used eight human breast cancer cell lines, which represent the major molecular subtypes of breast cancer, to validate the cytotoxic effects of MLN9708, alone and in combination with Dox. We found that MLN9708 had cytotoxic effects, induced autophagy and MKP-1 expression, and enhanced Dox-induced apoptosis in these cell lines. MLN9708 also enhanced Dox-induced JNK and p38 phosphorylation and inhibited Dox-induced IκBα degradation. Our in vitro results suggest that MLN9708 has antitumor effects in breast cancer and can sensitize breast cancer cells to Dox treatment. This promising combination may be an effective and feasible therapeutic option for treating breast cancer and warrants clinical validation. PMID:27217076

  5. The genetic basis for the biosynthesis of the pharmaceutically important class of epoxyketone proteasome inhibitors

    PubMed Central

    Schorn, Michelle; Zettler, Judith; Noel, Joseph P.; Dorrestein, Pieter C.; Moore, Bradley S.; Kaysser, Leonard

    2013-01-01

    The epoxyketone proteasome inhibitors are an established class of therapeutic agents for the treatment of cancer. Their unique α′,β′-epoxyketone pharmacophore allows binding to the catalytic β-subunits of the proteasome with extraordinary specificity. Here we report the characterization of the first gene clusters for the biosynthesis of natural peptidyl-epoxyketones. The clusters for epoxomicin, the lead compound for the anti-cancer drug Kyprolis™, and for eponemycin were identified in the actinobacterial producer strains ATCC 53904 and Streptomyces hygroscopicus ATCC 53709, respectively, using a modified protocol for Ion Torrent PGM genome sequencing. Both gene clusters code for a hybrid non-ribosomal peptide synthetase/polyketide synthase multifunctional enzyme complex and homologous redox enzymes. Epoxomicin and eponemycin were heterologously produced in Streptomyces albus J1046 via whole pathway expression. Moreover, we employed mass spectral molecular networking for a new comparative metabolomics approach in a heterologous system and discovered a number of putative epoxyketone derivatives. With this study we have definitively linked epoxyketone proteasome inhibitors and their biosynthesis genes for the first time in any organism, which will now allow for their detailed biochemical investigation. PMID:24168704

  6. Next-generation proteasome inhibitor MLN9708 sensitizes breast cancer cells to doxorubicin-induced apoptosis

    PubMed Central

    Wang, Hao; Yu, Yang; Jiang, Zheng; Cao, Wen-Ming; Wang, Zhenyu; Dou, Jun; Zhao, Yanling; Cui, Yunfu; Zhang, Hong

    2016-01-01

    Doxorubicin (Dox), one of the most effective chemotherapy drug for cancer treatment, is limited by its severe side effects and chemoresistance. Dox induces DNA damage and leads to significant proteomic changes in the cancer cells, which makes the ubiquitin-proteasome system a potential target to enhance the efficacy of Dox therapy. The unsuccessful clinical trials of proteasome inhibitor PS-341 (bortezomib) in solid tumors led to the invention of MLN9708 (ixazomib), an orally bioavailable next-generation proteasome inhibitor with improved pharmacokinetic and pharmacodynamic features. In this preclinical study, we used eight human breast cancer cell lines, which represent the major molecular subtypes of breast cancer, to validate the cytotoxic effects of MLN9708, alone and in combination with Dox. We found that MLN9708 had cytotoxic effects, induced autophagy and MKP-1 expression, and enhanced Dox-induced apoptosis in these cell lines. MLN9708 also enhanced Dox-induced JNK and p38 phosphorylation and inhibited Dox-induced IκBα degradation. Our in vitro results suggest that MLN9708 has antitumor effects in breast cancer and can sensitize breast cancer cells to Dox treatment. This promising combination may be an effective and feasible therapeutic option for treating breast cancer and warrants clinical validation. PMID:27217076

  7. LMP2-specific inhibitors: chemical genetic tools for proteasome biology.

    PubMed

    Ho, Yik Khuan; Bargagna-Mohan, Paola; Wehenkel, Marie; Mohan, Royce; Kim, Kyung-Bo

    2007-04-01

    The immunoproteasome, having been linked to neurodegenerative diseases and hematological cancers, has been shown to play an important role in MHC class I antigen presentation. However, its other pathophysiological functions are still not very well understood. This can be attributed mainly to a lack of appropriate molecular probes that can selectively modulate the immunoproteasome catalytic subunits. Herein, we report the development of molecular probes that selectively inhibit the major catalytic subunit, LMP2, of the immunoproteasome. We show that these compounds irreversibly modify the LMP2 subunit with high specificity. Importantly, LMP2-rich cancer cells compared to LMP2-deficient cancer cells are more sensitive to growth inhibition by the LMP2-specific inhibitor, implicating an important role of LMP2 in regulating cell growth of malignant tumors that highly express LMP2. PMID:17462577

  8. Proteasome inhibitor model of Parkinson's disease in mice is confounded by neurotoxicity of the ethanol vehicle.

    PubMed

    Landau, Anne M; Kouassi, Edouard; Siegrist-Johnstone, Rosmarie; Desbarats, Julie

    2007-02-15

    Defects in the ubiquitin-proteasome system have been implicated in Parkinson's Disease (PD). Recently, a rat model of PD was developed using a synthetic proteasome inhibitor (PSI), (Z-lle-Glu(OtBu)-Ala-Leu-al). We attempted to transfer this model to mouse studies, where genetics can be more readily investigated due to the availability of genetically modified mice. We treated C57BL/6 (B6) mice with six intraperitoneal injections of 6 mg/kg PSI in 50 mul of 70% ethanol over a 2-week-period. We found significant decreases in nigrostriatal dopamine in PSI-treated mice compared with saline-treated mice. However, we observed similar decreases in the ethanol-treated vehicle control group. Administration of ethanol alone led to significant long-term alterations in dopamine levels. Ethanol significantly eclipses the effects of PSI in the dopamine system, and therefore is a confounding vehicle for this model. PMID:17230468

  9. Proteasome Inhibitor YSY01A Enhances Cisplatin Cytotoxicity in Cisplatin-Resistant Human Ovarian Cancer Cells

    PubMed Central

    Huang, Wei; Zhou, Quan; Yuan, Xia; Ge, Ze-mei; Ran, Fu-xiang; Yang, Hua-yu; Qiang, Guang-liang; Li, Run-tao; Cui, Jing-rong

    2016-01-01

    Cisplatin is one of the most common drugs used for treatment of solid tumors such as ovarian cancer. Unfortunately, the development of resistance against this cytotoxic agent limits its clinical use. Here we report that YSY01A, a novel proteasome inhibitor, is capable of suppressing survival of cisplatin-resistant ovarian cancer cells by inducing apoptosis. And YSY01A treatment enhances the cytotoxicity of cisplatin in drug-resistant ovarian cancer cells. Specifically, YSY01A abrogates regulatory proteins important for cell proliferation and anti-apoptosis including NF-κB p65 and STAT3, resulting in down-regulation of Bcl-2. A dramatic increase in cisplatin uptake was also observed by inductively coupled plasma-mass spectrometry following exposure to YSY01A. Taken together, YSY01A serves as a potential candidate for further development as anticancer therapeutics targeting the proteasome. PMID:27326257

  10. Budding of Equine Infectious Anemia Virus Is Insensitive to Proteasome Inhibitors

    PubMed Central

    Patnaik, Akash; Chau, Vincent; Li, Feng; Montelaro, Ronald C.; Wills, John W.

    2002-01-01

    The only retrovirus protein required for the budding of virus-like particles is the Gag protein; however, recent studies of Rous sarcoma virus (RSV) and human immunodeficiency virus have suggested that modification of Gag with ubiquitin (Ub) is also required. As a consequence, the release of these viruses is reduced in the presence of proteasome inhibitors, which indirectly reduce the levels of free Ub within the cell. Here we show that the budding of equine infectious anemia virus (EIAV) from infected equine cells is largely unaffected by these drugs, although use of one inhibitor (MG-132) resulted in a dramatic block to proteolytic processing of Gag. This lack of sensitivity was also observed in transiently transfected avian cells under conditions that greatly reduce RSV budding. Moreover, insensitivity was observed when the EIAV Gag protein was expressed in the absence of all the other virus products, indicating that they are not required for this phenotype. An activity that enables EIAV to tolerate exposure to proteasome inhibitors was mapped to the C-terminal p9 sequence, as demonstrated by the ability of an RSV Gag-p9 chimera to bud in the presence of the drugs. Intriguingly, the p9 sequence contains a short sequence motif that is similar to a surface-exposed helix of Ub, suggesting that EIAV Gag may have captured a function that allows it to bypass the need for ubiquitination. Thus, the mechanism of EIAV budding may not be substantially different from that of other retroviruses, even though it behaves differently in the presence of proteasome inhibitors. PMID:11861830

  11. Two waves of proteasome-dependent protein degradation in the hippocampus are required for recognition memory consolidation.

    PubMed

    Figueiredo, Luciana S; Dornelles, Arethuza S; Petry, Fernanda S; Falavigna, Lucio; Dargél, Vinicius A; Köbe, Luiza M; Aguzzoli, Cristiano; Roesler, Rafael; Schröder, Nadja

    2015-04-01

    Healthy neuronal function and synaptic modification require a concert of synthesis and degradation of proteins. Increasing evidence indicates that protein turnover mediated by proteasome activity is involved in long-term synaptic plasticity and memory. However, its role in different phases of memory remains debated, and previous studies have not examined the possible requirement of protein degradation in recognition memory. Here, we show that the proteasome inhibitor, lactacystin (LAC), infused into the CA1 area of the hippocampus at two specific time points during consolidation, impairs 24-retention of memory for object recognition in rats. Administration of LAC after retrieval did not affect retention. These findings provide the first evidence for a requirement of proteasome activity in recognition memory, indicate that protein degradation in the hippocampus is necessary during selective time windows of memory consolidation, and further our understanding of the role of protein turnover in memory formation. PMID:25687693

  12. Mitochondrial Bax translocation partially mediates synergistic cytotoxicity between histone deacetylase inhibitors and proteasome inhibitors in glioma cells

    PubMed Central

    Yu, Chunrong; Friday, Bret B.; Yang, Lin; Atadja, Peter; Wigle, Dennis; Sarkaria, Jann; Adjei, Alex A.

    2008-01-01

    The effects of combining histone deacetylase (HDAC) inhibitors and proteasome inhibitors were evaluated in both established glioblastoma multiforme (GBM) cell lines and short-term cultures derived from the Mayo Clinic xenograft GBM panel. Coexposure of LBH589 and bortezomib at minimally toxic doses of either drug alone resulted in a striking induction of apoptosis in established U251, U87, and D37 GBM cell lines, as well as in GBM8, GBM10, GBM12, GBM14, and GBM56 short-term cultured cell lines. Synergism of apoptosis induction was also observed in U251 cells when coexposing cells to other HDAC inhibitors, including LAQ824 and trichostatin A, with the proteasome inhibitor MG132, thus demonstrating a class effect. In U251 cells, bortezomib alone or in combination with LBH589 decreased Raf-1 levels and suppressed Akt and Erk activation. LBH589 or bortezomib alone increased expression of the cell cycle regulators p21 and p27. Additionally, the combination, but not the individual agents, markedly enhanced JNK activation. Synergistic induction of apoptosis after exposure to LBH589 and bortezomib was partially mediated by Bax translocation from the cytosol to the mitochondria resulting from Bax conformational changes. Bax translocation precedes cytochrome c release and apoptosis, and selective down-regulation of Bax using siRNA significantly mitigates the cytotoxicity of LBH589 and bortezomib. This combination regimen warrants further preclinical and possible clinical study for glioma patients. PMID:18445700

  13. Combination of proteasome inhibitors bortezomib and NPI-0052 trigger in vivo synergistic cytotoxicity in multiple myeloma.

    PubMed

    Chauhan, Dharminder; Singh, Ajita; Brahmandam, Mohan; Podar, Klaus; Hideshima, Teru; Richardson, Paul; Munshi, Nikhil; Palladino, Michael A; Anderson, Kenneth C

    2008-02-01

    Our recent study demonstrated that a novel proteasome inhibitor NPI-0052 triggers apoptosis in multiple myeloma (MM) cells, and importantly, that is distinct from bortezomib (Velcade) in its chemical structure, effects on proteasome activities, and mechanisms of action. Here, we demonstrate that combining NPI-0052 and bortezomb induces synergistic anti-MM activity both in vitro using MM cell lines or patient CD138(+) MM cells and in vivo in a human plasmacytoma xenograft mouse model. NPI-0052 plus bortezomib-induced synergistic apoptosis is associated with: (1) activation of caspase-8, caspase-9, caspase-3, and PARP; (2) induction of endoplasmic reticulum (ER) stress response and JNK; (3) inhibition of migration of MM cells and angiogenesis; (4) suppression of chymotrypsin-like (CT-L), caspase-like (C-L), and trypsin-like (T-L) proteolytic activities; and (5) blockade of NF-kappaB signaling. Studies in a xenograft model show that low dose combination of NPI-0052 and bortezomib is well tolerated and triggers synergistic inhibition of tumor growth and CT-L, C-L, and T-L proteasome activities in tumor cells. Immununostaining of MM tumors from NPI-0052 plus bortezomib-treated mice showed growth inhibition, apoptosis, and a decrease in associated angiogenesis. Taken together, our study provides the preclinical rationale for clinical protocols evaluating bortezomib together with NPI-0052 to improve patient outcome in MM. PMID:18006697

  14. Structure-Based Design of β5c Selective Inhibitors of Human Constitutive Proteasomes.

    PubMed

    Xin, Bo-Tao; de Bruin, Gerjan; Huber, Eva M; Besse, Andrej; Florea, Bogdan I; Filippov, Dmitri V; van der Marel, Gijsbert A; Kisselev, Alexei F; van der Stelt, Mario; Driessen, Christoph; Groll, Michael; Overkleeft, Herman S

    2016-08-11

    This work reports the development of highly potent and selective inhibitors of the β5c catalytic activity of human constitutive proteasomes. The work describes the design principles, large hydrophobic P3 residue and small hydrophobic P1 residue, that led to the synthesis of a panel of peptide epoxyketones; their evaluation and the selection of the most promising compounds for further analyses. Structure-activity relationships detail how in a logical order the β1c/i, β2c/i, and β5i activities became resistant to inhibition as compounds were diversified stepwise. The most effective compounds were obtained as a mixture of cis- and trans-biscyclohexyl isomers, and enantioselective synthesis resolved this issue. Studies on yeast proteasome structures complexed with some of the compounds provide a rationale for the potency and specificity. Substitution of the N-terminus in the most potent compound for a more soluble equivalent led to a cell-permeable molecule that selectively and efficiently blocks β5c in cells expressing both constitutive proteasomes and immunoproteasomes. PMID:27438186

  15. Partial proteasome inhibitors induce hair follicle growth by stabilizing β-catenin.

    PubMed

    Yucel, Gozde; Van Arnam, John; Means, Paula Casey; Huntzicker, Erik; Altindag, Banu; Lara, Maria Fernanda; Yuan, Jenny; Kuo, Calvin; Oro, Anthony E

    2014-01-01

    The activation of tissue stem cells from their quiescent state represents the initial step in the complex process of organ regeneration and tissue repair. While the identity and location of tissue stem cells are becoming known, how key regulators control the balance of activation and quiescence remains mysterious. The vertebrate hair is an ideal model system where hair cycling between growth and resting phases is precisely regulated by morphogen signaling pathways, but how these events are coordinated to promote orderly signaling in a spatial and temporal manner remains unclear. Here, we show that hair cycle timing depends on regulated stability of signaling substrates by the ubiquitin-proteasome system. Topical application of partial proteasomal inhibitors (PaPIs) inhibits epidermal and dermal proteasome activity throughout the hair cycle. PaPIs prevent the destruction of the key anagen signal β-catenin, resulting in more rapid hair growth and dramatically shortened telogen. We show that PaPIs induce excess β-catenin, act similarly to the GSK3β antagonist LiCl, and antagonize Dickopf-related protein-mediated inhibition of anagen. PaPIs thus represent a novel class of hair growth agents that act through transiently modifying the balance of stem cell activation and quiescence pathways. PMID:23963711

  16. Partial Proteasome Inhibitors Induce Hair Follicle Growth by Stabilizing β-catenin

    PubMed Central

    Yucel, Gozde; Van Arnam, John; Means, Paula Casey; Huntzicker, Erik; Altindag, Banu; Lara, Maria Fernanda; Yuan, Jenny; Kuo, Calvin; Oro, Anthony E.

    2014-01-01

    The activation of tissue stem cells from their quiescent state represents the initial step in the complex process of organ regeneration and tissue repair. While the identity and location of tissue stem cells are becoming known, how key regulators control the balance of activation and quiescence remains mysterious. The vertebrate hair is an ideal model system where hair cycling between growth and resting phases is precisely regulated by morphogen signaling pathways, but how these events are coordinated to promote orderly signaling in a spatial and temporal manner remains unclear. Here, we show that hair cycle timing depends on regulated stability of signaling substrates by the ubiquitin-proteasome system. Topical application of partial proteasomal inhibitors (PaPIs) inhibits epidermal and dermal proteasome activity throughout the hair cycle. PaPIs prevent the destruction of the key anagen signal β-catenin, resulting in more rapid hair growth and dramati cally shortened telogen. We show that PaPIs induce excess β-catenin, act similarly to the GSK3β antagonist LiCl, and antagonize Dickopf-related protein-mediated inhibition of anagen. PaPIs thus represent a novel class of hair growth agents that act through transiently modifying the balance of stem cell activation and quiescence pathways. PMID:23963711

  17. A potent and selective inhibitor for the UBLCP1 proteasome phosphatase

    PubMed Central

    He, Yantao; Guo, Xing; Yu, Zhi-Hong; Wu, Li; Gunawan, Andrea M.; Zhang, Yan; Dixon, Jack E.; Zhang, Zhong-Yin

    2015-01-01

    The ubiquitin-like domain-containing C-terminal domain phosphatase 1 (UBLCP1) has been implicated as a negative regulator of the proteasome, a key mediator in the ubiquitin-dependent protein degradation. Small molecule inhibitors that block UBLCP1 activity would be valuable as research tools and potential therapeutics for human diseases caused by the cellular accumulation of misfold/damaged proteins. We report a salicylic acid fragment-based library approach aimed at targeting both the phosphatase active site and its adjacent binding pocket for enhanced affinity and selectivity. Screening of the focused libraries led to the identification of the first potent and selective UBLCP1 inhibitor 13. Compound 13 exhibits an IC50 of 1.0 μM for UBLCP1 and greater than 5-fold selectivity against a large panel of protein phosphatases from several distinct families. Importantly, the inhibitor possesses efficacious cellular activity and is capable of inhibiting UBLCP1 function in cells, which in turn up-regulates nuclear proteasome activity. These studies set the groundwork for further developing compound 13 into chemical probes or potential therapeutic agents targeting the UBLCP1 phosphatase. PMID:25907364

  18. Disulfiram promotes the conversion of carcinogenic cadmium to a proteasome inhibitor with pro-apoptotic activity in human cancer cells

    SciTech Connect

    Li Lihua; Yang Huanjie; Chen Di; Cui, Cindy; Ping Dou, Q.

    2008-06-01

    The ubiquitin-proteasome system is involved in various cellular processes, including transcription, apoptosis, and cell cycle. In vitro, in vivo, and clinical studies suggest the potential use of proteasome inhibitors as anticancer drugs. Cadmium (Cd) is a widespread environmental pollutant that has been classified as a human carcinogen. Recent study in our laboratory suggested that the clinically used anti-alcoholism drug disulfiram (DSF) could form a complex with tumor cellular copper, resulting in inhibition of the proteasomal chymotrypsin-like activity and induction of cancer cell apoptosis. In the current study, we report, for the first time, that DSF is able to convert the carcinogen Cd to a proteasome-inhibitor and cancer cell apoptosis inducer. Although the DSF-Cd complex inhibited the chymotrypsin-like activity of a purified 20S proteasome with an IC{sub 50} value of 32 {mu}mol/L, this complex was much more potent in inhibiting the chymotrypsin-like activity of prostate cancer cellular 26S proteasome. Inhibition of cellular proteasome activity by the DSF-Cd complex resulted in the accumulation of ubiquitinated proteins and the natural proteasome substrate p27, which was followed by activation of calpain and induction of apoptosis. Importantly, human breast cancer MCF10DCIS cells were much more sensitive to the DSF-Cd treatment than immortalized but non-tumorigenic human breast MCF-10A cells, demonstrating that the DSF-Cd complex could selectively induce proteasome inhibition and apoptosis in human tumor cells. Our work suggests the potential use of DSF for treatment of cells with accumulated levels of carcinogen Cd.

  19. Synthesis and Evaluation of Macrocyclic Peptide Aldehydes as Potent and Selective Inhibitors of the 20S Proteasome.

    PubMed

    Wilson, David L; Meininger, Isabel; Strater, Zack; Steiner, Stephanie; Tomlin, Frederick; Wu, Julia; Jamali, Haya; Krappmann, Daniel; Götz, Marion G

    2016-03-10

    This research explores the first design and synthesis of macrocyclic peptide aldehydes as potent inhibitors of the 20S proteasome. Two novel macrocyclic peptide aldehydes based on the ring-size of the macrocyclic natural product TMC-95 were prepared and evaluated as inhibitors of the 20S proteasome. Both compounds inhibited in the low nanomolar range and proved to be selective for the proteasome over other serine and cysteine proteases, particularly when compared to linear analogues with similar amino acid sequences. In HeLa cells, both macrocycles efficiently inhibited activation of nuclear factor-κB (NF-κB) transcription factor by blocking proteasomal degradation of the inhibitor protein IκBα after cytokine stimulation. Due to their covalent mechanism of binding these compounds represent a 1000-fold increase in inhibitory potency over previously reported noncovalently binding TMC-95 analogues. Molecular modeling of the macrocyclic peptides confirms the preference of the large S3 pocket for large, hydrophobic residues and the ability to exploit this to improve selectivity of proteasome inhibitors. PMID:26985310

  20. Oxadiazole-isopropylamides as Potent and Non-covalent Proteasome Inhibitors

    PubMed Central

    Ozcan, Sevil; Kazi, Aslamuzzaman; Marsilio, Frank; Fang, Bin; Guida, Wayne C.; Koomen, John; Lawrence, Harshani R.; Sebti, Saïd M.

    2013-01-01

    Screening of the 50,000 ChemBridge compound library led to the identification of the oxadiazole-isopropylamide 1 (PI-1833) which inhibited CT-L activity (IC50 0.60 μM) with little effects on the other 2 major proteasome proteolytic activities, T-L and PGPH-L. LC/MS-MS and dialysis show that 1 is a non-covalent and rapidly reversible CT-L inhibitor. Focused library synthesis provided 11ad (PI-1840) with CT-L activity (IC50 27 nM). Detailed SAR studies indicate that the amide moiety and the 2 phenyl rings are sensitive toward modifications. Hydrophobic residues, such as propyl or butyl, in the para-position (not ortho or meta) of the A-ring and a meta-pyridyl group as B-ring significantly improve activity. Compound 11ad (IC50 0.37 μM) is more potent than 1 (IC50 3.5 μM) at inhibiting CT-L activity in intact MDA-MB-468 human breast cancer cells and inhibiting their survival. The activity of 11ad warrants further pre-clinical investigation of this class as non-covalent proteasome inhibitors. PMID:23547706

  1. Multiplexed metagenome mining using short DNA sequence tags facilitates targeted discovery of epoxyketone proteasome inhibitors

    PubMed Central

    Owen, Jeremy G.; Charlop-Powers, Zachary; Smith, Alexandra G.; Ternei, Melinda A.; Calle, Paula Y.; Reddy, Boojala Vijay B.; Montiel, Daniel; Brady, Sean F.

    2015-01-01

    In molecular evolutionary analyses, short DNA sequences are used to infer phylogenetic relationships among species. Here we apply this principle to the study of bacterial biosynthesis, enabling the targeted isolation of previously unidentified natural products directly from complex metagenomes. Our approach uses short natural product sequence tags derived from conserved biosynthetic motifs to profile biosynthetic diversity in the environment and then guide the recovery of gene clusters from metagenomic libraries. The methodology is conceptually simple, requires only a small investment in sequencing, and is not computationally demanding. To demonstrate the power of this approach to natural product discovery we conducted a computational search for epoxyketone proteasome inhibitors within 185 globally distributed soil metagenomes. This led to the identification of 99 unique epoxyketone sequence tags, falling into 6 phylogenetically distinct clades. Complete gene clusters associated with nine unique tags were recovered from four saturating soil metagenomic libraries. Using heterologous expression methodologies, seven potent epoxyketone proteasome inhibitors (clarepoxcins A–E and landepoxcins A and B) were produced from these pathways, including compounds with different warhead structures and a naturally occurring halohydrin prodrug. This study provides a template for the targeted expansion of bacterially derived natural products using the global metagenome. PMID:25831524

  2. The class-I HDAC inhibitor MGCD0103 induces apoptosis in Hodgkin lymphoma cell lines and synergizes with proteasome inhibitors by an HDAC6-independent mechanism

    PubMed Central

    Buglio, Daniela; Mamidipudi, Vidya; Khaskhely, Noor M.; Brady, Helen; Heise, Carla; Besterman, Jeffrey; Martell, Robert E.; MacBeth, Kyle; Younes, Anas

    2011-01-01

    Summary Inhibition of histone deacetylase 6 (HDAC6)-dependent aggresome function by pan HDAC inhibitors was recently reported to be a key mechanism underlying the synergistic activity between proteasome inhibitors and HDAC inhibitors in a variety of tumour types. Because these combinations induce significant thrombocytopenia in vivo, we examined whether less toxic, isotype-selective HDAC inhibitors may still synergize with proteasome inhibitors, and if so, by what mechanisms. Here, we showed that the class I HDAC inhibitor, MGCD0103, has a potent antiproliferative activity in Hodgkin lymphoma (HL) cell lines. Furthermore, MGCD0103 induced tumour necrosis factor α (TNF-α) expression and secretion, which was associated with nuclear factor (NF)-κB activation. Selective inhibition of TNF- α expression by short interfering mRNA, or inhibition of MGCD0103-induced NF-kB activation by proteasome inhibitors enhanced MGCD0103-induced cell death. Thus, our results demonstrate that MGCD0103 may synergize with proteasome inhibitors by HDAC6-independent mechanisms, providing mechanistic rationale for exploring this potentially less toxic combination for the treatment of lymphoma. PMID:20880107

  3. A reversible and highly selective inhibitor of the proteasomal ubiquitin receptor rpn13 is toxic to multiple myeloma cells.

    PubMed

    Trader, Darci J; Simanski, Scott; Kodadek, Thomas

    2015-05-20

    The proteasome is a multisubunit complex responsible for most nonlysosomal turnover of proteins in eukaryotic cells. Proteasome inhibitors are of great interest clinically, particularly for the treatment of multiple myeloma (MM). Unfortunately, resistance arises almost inevitably to these active site-targeted drugs. One strategy to overcome this resistance is to inhibit other steps in the protein turnover cascade mediated by the proteasome. Previously, Anchoori et al. identified Rpn13 as the target of an electrophilic compound (RA-190) that was selectively toxic to MM cells (Cancer Cell 2013, 24, 791-805), suggesting that this subunit of the proteasome is also a viable cancer drug target. Here we describe the discovery of the first highly selective, reversible Rpn13 ligands and show that they are also selectively toxic to MM cells. These data strongly support the hypothesis that Rpn13 is a viable target for the development of drugs to treat MM and other cancers. PMID:25914958

  4. Disulfiram promotes the conversion of carcinogenic cadmium to a proteasome inhibitor with pro-apoptotic activity in human cancer cells

    PubMed Central

    Li, Lihua; Yang, Huanjie; Chen, Di; Cui, Cindy; Dou, Q. Ping

    2013-01-01

    The ubiquitinproteasome system is involved in various cellular processes, including transcription, apoptosis, and cell cycle. In vitro, in vivo, and clinical studies suggest the potential use of proteasome inhibitors as anticancer drugs. Cadmium (Cd) is a widespread environmental pollutant that has been classified as a human carcinogen. Recent study in our laboratory suggested that the clinically used anti-alcoholism drug disulfiram (DSF) could form a complex with tumor cellular copper, resulting in inhibition of the proteasomal chymotrypsin-like activity and induction of cancer cell apoptosis. In the current study, we report, for the first time, that DSF is able to convert the carcinogen Cd to a proteasome-inhibitor and cancer cell apoptosis inducer. Although the DSF–Cd complex inhibited the chymotrypsin-like activity of a purified 20S proteasome with an IC50 value of 32 μmol/L, this complex was much more potent in inhibiting the chymotrypsin-like activity of prostate cancer cellular 26S proteasome. Inhibition of cellular proteasome activity by the DSF–Cd complex resulted in the accumulation of ubiquitinated proteins and the natural proteasome substrate p27, which was followed by activation of calpain and induction of apoptosis. Importantly, human breast cancer MCF10DCIS cells were much more sensitive to the DSF–Cd treatment than immortalized but non-tumorigenic human breast MCF-10A cells, demonstrating that the DSF–Cd complex could selectively induce proteasome inhibition and apoptosis in human tumor cells. Our work suggests the potential use of DSF for treatment of cells with accumulated levels of carcinogen Cd. PMID:18304598

  5. Histone deacetylase inhibitor trichostatin A and proteasome inhibitor PS-341 synergistically induce apoptosis in pancreatic cancer cells

    SciTech Connect

    Bai Jirong . E-mail: jbai@bidmc.harvard.edu; Demirjian, Aram; Sui Jianhua; Marasco, Wayne; Callery, Mark P. . E-mail: mcallery@bidmc.harvard.ede

    2006-10-06

    Pancreatic cancer is a common and lethal malignancy. Pancreatic cancer cells overexpress multiple anti-apoptotic factors and death receptor decoys, and are strongly resistant to radiation and to 5-fluorouracil (5-FU)- or gemcitabine (Gem)-based chemotherapy regimens. We have found that low-dose proteasome inhibitor PS-341 and histone deacetylase inhibitor trichostatin A (TSA) synergistically induce cytotoxicity in a panel of eight diverse pancreatic cancer cell lines. Combining TSA with PS-341 effectively inactivated NF{kappa}B signaling, downregulated the predominant endogenous anti-apoptotic factor Bcl-XL overexpression, and disrupted MAP kinase pathway. The combined drug regimen effectively inflicted an average of 71.5% apoptotic cell death (55.2-80%) in diverse pancreatic cancer cell lines by activating the intrinsic apoptotic pathway. Conclusion: the TSA/PS-341 regimen may represent a potential novel therapeutic strategy for pancreatic cancer.

  6. Enhanced efficacy against cervical carcinomas through polymeric micelles physically incorporating the proteasome inhibitor MG132.

    PubMed

    Matsumoto, Yoko; Miyamoto, Yuichiro; Cabral, Horacio; Matsumoto, Yu; Nagasaka, Kazunori; Nakagawa, Shunsuke; Yano, Tetsu; Maeda, Daichi; Oda, Katsutoshi; Kawana, Kei; Nishiyama, Nobuhiro; Kataoka, Kazunori; Fujii, Tomoyuki

    2016-06-01

    Treatment of recurrent or advanced cervical cancer is still limited, and new therapeutic choices are needed for improving prognosis and quality of life of patients. Because human papilloma virus (HPV) infection is critical in cervical carcinogenesis, with the E6 and E7 oncogenes of HPV degrading tumor suppressor proteins through the ubiquitin proteasome system, the inhibition of the ubiquitin proteasome system appears to be an ideal target to suppress the growth of cervical tumors. Herein, we focused on the ubiquitin proteasome inhibitor MG132 (carbobenzoxy-Leu-Leu-leucinal) as an anticancer agent against cervical cancer cells, and physically incorporated it into micellar nanomedicines for achieving selective delivery to solid tumors and improving its in vivo efficacy. These MG132-loaded polymeric micelles (MG132/m) showed strong tumor inhibitory in vivo effect against HPV-positive tumors from HeLa and CaSki cells, and even in HPV-negative tumors from C33A cells. Repeated injection of MG132/m showed no significant toxicity to mice under analysis by weight change or histopathology. Moreover, the tumors treated with MG132/m showed higher levels of tumor suppressing proteins, hScrib and p53, as well as apoptotic degree, than tumors treated with free MG132. This enhanced efficacy of MG132/m was attributed to their prolonged circulation in the bloodstream, which allowed their gradual extravasation and penetration within the tumor tissue, as determined by intravital microscopy. These results support the use of MG132 incorporated into polymeric micelles as a safe and effective therapeutic strategy against cervical tumors. PMID:26987571

  7. Proteasomal Inhibition Restores Biological Function of Mis-sense Mutated Dysferlin in Patient-derived Muscle Cells*

    PubMed Central

    Azakir, Bilal A.; Di Fulvio, Sabrina; Kinter, Jochen; Sinnreich, Michael

    2012-01-01

    Dysferlin is a transmembrane protein implicated in surface membrane repair of muscle cells. Mutations in dysferlin cause the progressive muscular dystrophies Miyoshi myopathy, limb girdle muscular dystrophy 2B, and distal anterior compartment myopathy. Dysferlinopathies are inherited in an autosomal recessive manner, and many patients with this disease harbor mis-sense mutations in at least one of their two pathogenic DYSF alleles. These patients have significantly reduced or absent dysferlin levels in skeletal muscle, suggesting that dysferlin encoded by mis-sense alleles is rapidly degraded by the cellular quality control system. We reasoned that mis-sense mutated dysferlin, if salvaged from degradation, might be biologically functional. We used a dysferlin-deficient human myoblast culture harboring the common R555W mis-sense allele and a DYSF-null allele, as well as control human myoblast cultures harboring either two wild-type or two null alleles. We measured dysferlin protein and mRNA levels, resealing kinetics of laser-induced plasmalemmal wounds, myotube formation, and cellular viability after treatment of the human myoblast cultures with the proteasome inhibitors lactacystin or bortezomib (Velcade). We show that endogenous R555W mis-sense mutated dysferlin is degraded by the proteasomal system. Inhibition of the proteasome by lactacystin or Velcade increases the levels of R555W mis-sense mutated dysferlin. This salvaged protein is functional as it restores plasma membrane resealing in patient-derived myoblasts and reverses their deficit in myotube formation. Bortezomib and lactacystin did not cause cellular toxicity at the regimen used. Our results raise the possibility that inhibition of the degradation pathway of mis-sense mutated dysferlin could be used as a therapeutic strategy for patients harboring certain dysferlin mis-sense mutations. PMID:22318734

  8. Cytoplasmic Trafficking of Minute Virus of Mice: Low-pH Requirement, Routing to Late Endosomes, and Proteasome Interaction

    PubMed Central

    Ros, Carlos; Burckhardt, Christoph J.; Kempf, Christoph

    2002-01-01

    The cytoplasmic trafficking of the prototype strain of minute virus of mice (MVMp) was investigated by analyzing and quantifying the effect of drugs that reduce or abolish specific cellular functions on the accumulation of viral macromolecules. With this strategy, it was found that a low endosomal pH is required for the infection, since bafilomycin A1 and chloroquine, two pH-interfering drugs, were similarly active against MVMp. Disruption of the endosomal network by brefeldin A interfered with MVMp infection, indicating that viral particles are routed farther than the early endocytic compartment. Pulse experiments with endosome-interfering drugs showed that the bulk of MVMp particles remained in the endosomal compartment for several hours before its release to the cytosol. Drugs that block the activity of the proteasome by different mechanisms, such as MG132, lactacystin, and epoxomicin, all strongly blocked MVMp infection. Pulse experiments with the proteasome inhibitor MG132 indicated that MVMp interacts with cellular proteasomes after endosomal escape. The chymotrypsin-like but not the trypsin-like activity of the proteasome is required for the infection, since the chymotrypsin inhibitors N-tosyl-l-phenylalanine chloromethyl ketone and aclarubicin were both effective in blocking MVMp infection. However, the trypsin inhibitor Nα-p-tosyl-l-lysine chloromethyl ketone had no effect. These results suggest that the ubiquitin-proteasome pathway plays an essential role in the MVMp life cycle, probably assisting at the stages of capsid disassembly and/or nuclear translocation. PMID:12438589

  9. THE BTK INHIBITOR PCI-32765 SYNERGISTICALLY INCREASES PROTEASOME INHIBITOR ACTIVITY IN DLBCL AND MCL CELLS SENSITIVE OR RESISTANT TO BORTEZOMIB

    PubMed Central

    Dasmahapatra, Girija; Patel, Hiral; Dent, Paul; Fisher, Richard I.; Friedberg, Jonathan; Grant, Steven

    2012-01-01

    Summary Interactions between the Bruton tyrosine kinase (BTK) inhibitor PCI-32765 and the proteasome inhibitor (bortezomib) were examined in diffuse large-B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) cells, including those highly resistant to bortezomib. Co-administration of PCI-32765/bortezomib synergistically increased mitochondrial injury and apoptosis in germinal centre- or activated B-cell-like-DLBCL cells and in MCL cells. These events were accompanied by marked AKT and nuclear factor (NF)-κB (NFKB1) inactivation, down-regulation of Mcl-1 (MCL1), Bcl-xL (BCL2L1), and XIAP, and enhanced DNA damage (e.g., γH2A.X formation) and endoplasmic reticulum (ER) stress. Similar interactions were observed in highly bortezomib-resistant DLBCL and MCL cells, and in primary DLBCL cells. In contrast, PCI-32765/bortezomib regimens displayed minimal toxicity toward normal CD34+ bone marrow cells. Transfection of DLBCL cells with a constitutively active AKT construct attenuated AKT inactivation and significantly diminished cell death, whereas expression of an NF-κB “super-repressor” (IκBαser34/36) increased both PCI-32765 and bortezomib lethality. Moreover, cells in which the ER stress response was disabled by a dominant-negative eIF2α construct were resistant to this regimen. Finally, combined exposure to PCI-32765 and bortezomib resulted in more pronounced and sustained reactive oxygen species (ROS) generation, and ROS scavengers significantly diminished lethality. Given promising early clinical results for PCI-32765 in DLBCL and MCL, a strategy combining BTK/ proteasome inhibitor warrants attention in these malignancies. PMID:23360303

  10. Why does threonine, and not serine, function as the active site nucleophile in proteasomes?

    PubMed

    Kisselev, A F; Songyang, Z; Goldberg, A L

    2000-05-19

    Proteasomes belong to the N-terminal nucleophile group of amidases and function through a novel proteolytic mechanism, in which the hydroxyl group of the N-terminal threonines is the catalytic nucleophile. However, it is unclear why threonine has been conserved in all proteasomal active sites, because its replacement by a serine in proteasomes from the archaeon Thermoplasma acidophilum (T1S mutant) does not alter the rates of hydrolysis of Suc-LLVY-amc (Seemüller, E., Lupas, A., Stock, D., Lowe, J., Huber, R., and Baumeister, W. (1995) Science 268, 579-582) and other standard peptide amide substrates. However, we found that true peptide bonds in decapeptide libraries were cleaved by the T1S mutant 10-fold slower than by wild type (wt) proteasomes. In degrading proteins, the T1S proteasome was 3.5- to 6-fold slower than the wt, and this difference increased when proteolysis was stimulated using the proteasome-activating nucleotidase (PAN) ATPase complex. With mutant proteasomes, peptide bond cleavage appeared to be rate-limiting in protein breakdown, unlike with wt. Surprisingly, a peptide ester was hydrolyzed by both particles much faster than the corresponding amide, and the T1S mutant cleaved it faster than the wt. Moreover, the T1S mutant was inactivated by the ester inhibitor clasto-lactacystin-beta-lactone severalfold faster than the wt, but reacted with nonester irreversible inhibitors at similar rates. T1A and T1C mutants were completely inactive in all these assays. Thus, proteasomes lack additional active sites, and the N-terminal threonine evolved because it allows more efficient protein breakdown than serine. PMID:10809725

  11. Salinosporamide Natural Products: Potent 20S Proteasome Inhibitors as Promising Cancer Chemotherapeutics

    PubMed Central

    Gulder, Tobias A. M.

    2010-01-01

    Proteasome inhibitors are rapidly evolving as potent treatment options in cancer therapy. One of the most promising drug candidates of this type is salinosporamide A from the bacterium Salinispora tropica. This marine natural product possesses a complex, densely functionalized γ-lactam-β-lactone pharmacophore, which is responsible for its irreversible binding to its target, the β subunit of the 20S proteasome. Salinosporamide A entered phase I clinical trials for the treatment of multiple myeloma only three years after its discovery. The strong biological activity and the challenging structure of this compound have fueled intense academic and industrial research in recent years, which has led to the development of more than ten syntheses, the elucidation of its biosynthetic pathway, and the generation of promising structure–activity relationships and oncological data. Salinosporamide A thus serves as an intriguing example of the successful interplay of modern drug discovery and biomedical research, medicinal chemistry and pharmacology, natural product synthesis and analysis, as well as biosynthesis and bioengineering. PMID:20927786

  12. Local application of a proteasome inhibitor enhances fracture healing in rats.

    PubMed

    Yoshii, Toshitaka; Nyman, Jeffry S; Yuasa, Masato; Esparza, Javier M; Okawa, Atsushi; Gutierrez, Gloria E

    2015-08-01

    The ubiquitin/proteasome system plays an important role in regulating the activity of osteoblast precursor cells. Proteasome inhibitors (PSIs) have been shown to stimulate the differentiation of osteoblast precursor cells and to promote bone formation. This raises the possibility that PSIs might be useful for enhancing fracture healing. In this study, we examined the effect of the local administration of PSI on fracture repair in rats. The effects of treatment on the healing of a fractured femur were assessed based on radiographs, micro-computed tomography (μCT) analysis, biomechanical testing, and histological analysis. PSI enhanced osteogenic differentiation in bone marrow- and periosteum-derived mesenchymal progenitor cells in vitro. Moreover, the local administration of PSI in vivo promoted fracture healing in rats, as demonstrated by an increased fracture callus volume in radiographs at 2 weeks post-fracture, and improved radiographic scores. By week 4, PSI treatment had enhanced biomechanical strength and mineral density in the callus as assessed using bending tests, and μCT, respectively. Histological sections demonstrated that PSI treatment accelerated endochondral ossification during the early stages of fracture repair. Although further investigations are necessary to assess its clinical use, the local administration of PSIs might be a novel, and effective therapeutic approach for fracture repair. PMID:25683968

  13. A novel proteasome inhibitor acting in mitochondrial dysfunction, ER stress and ROS production.

    PubMed

    Maria, Durvanei Augusto; de Souza, Jean Gabriel; Morais, Katia L P; Berra, Carolina Maria; Zampolli, Hamilton de Campos; Demasi, Marilene; Simons, Simone Michaela; de Freitas Saito, Renata; Chammas, Roger; Chudzinski-Tavassi, Ana Marisa

    2013-06-01

    In cancer-treatment, potentially therapeutic drugs trigger their effects through apoptotic mechanisms. Generally, cell response is manifested by Bcl-2 family protein regulation, the impairment of mitochondrial functions, and ROS production. Notwithstanding, several drugs operate through proteasome inhibition, which, by inducing the accumulation and aggregation of misfolded or unfolded proteins, can lead to endoplasmic reticulum (ER) stress. Accordingly, it was shown that Amblyomin-X, a Kunitz-type inhibitor identified in the transcriptome of the Amblyomma cajennense tick by ESTs sequence analysis of a cDNA library, obtained in recombinant protein form, induces apoptosis in murine renal adenocarcinoma (RENCA) cells by: inducing imbalance between pro- and anti-apoptotic Bcl-2 family proteins, dysfunction/mitochondrial damage, production of reactive oxygen species (ROS), caspase cascade activation, and proteasome inhibition, all ER-stress inductive. Moreover, there was no manifest action on normal mouse-fibroblast cells (NHI3T3), suggesting an Amblyomin-X tumor-cell selectivity. Taken together, these evidences indicate that Amblyomin-X could be a promising candidate for cancer therapy. PMID:22975862

  14. Withaferin A: a proteasomal inhibitor promotes healing after injury and exerts anabolic effect on osteoporotic bone.

    PubMed

    Khedgikar, V; Kushwaha, P; Gautam, J; Verma, A; Changkija, B; Kumar, A; Sharma, S; Nagar, G K; Singh, D; Trivedi, P K; Sangwan, N S; Mishra, P R; Trivedi, R

    2013-01-01

    Withania somnifera or Ashwagandha is a medicinal herb of Ayurveda. Though the extract and purified molecules, withanolides, from this plant have been shown to have different pharmacological activities, their effect on bone formation has not been studied. Here, we show that one of the withanolide, withaferin A (WFA) acts as a proteasomal inhibitor (PI) and binds to specific catalytic β subunit of the 20S proteasome. It exerts positive effect on osteoblast by increasing osteoblast proliferation and differentiation. WFA increased expression of osteoblast-specific transcription factor and mineralizing genes, promoted osteoblast survival and suppressed inflammatory cytokines. In osteoclast, WFA treatment decreased osteoclast number directly by decreasing expression of tartarate-resistant acid phosphatase and receptor activator of nuclear factor kappa-B (RANK) and indirectly by decreasing osteoprotegrin/RANK ligand ratio. Our data show that in vitro treatment of WFA to calvarial osteoblast cells decreased expression of E3 ubiquitin ligase, Smad ubiquitin regulatory factor 2 (Smurf2), preventing degradation of Runt-related transcription factor 2 (RunX2) and relevant Smad proteins, which are phosphorylated by bone morphogenetic protein 2. Increased Smurf2 expression due to exogenous treatment of tumor necrosis factor α (TNFα) to primary osteoblast cells was decreased by WFA treatment. This was corroborated by using small interfering RNA against Smurf2. Further, WFA also blocked nuclear factor kappa-B (NF-kB) signaling as assessed by tumor necrosis factor stimulated nuclear translocation of p65-subunit of NF-kB. Overall data show that in vitro proteasome inhibition by WFA simultaneously promoted osteoblastogenesis by stabilizing RunX2 and suppressed osteoclast differentiation, by inhibiting osteoclastogenesis. Oral administration of WFA to osteopenic ovariectomized mice increased osteoprogenitor cells in the bone marrow and increased expression of osteogenic genes. WFA

  15. The Proteasome Inhibitor Bortezomib Maintains Osteocyte Viability in Multiple Myeloma Patients by Reducing Both Apoptosis and Autophagy: A New Function for Proteasome Inhibitors.

    PubMed

    Toscani, Denise; Palumbo, Carla; Dalla Palma, Benedetta; Ferretti, Marzia; Bolzoni, Marina; Marchica, Valentina; Sena, Paola; Martella, Eugenia; Mancini, Cristina; Ferri, Valentina; Costa, Federica; Accardi, Fabrizio; Craviotto, Luisa; Aversa, Franco; Giuliani, Nicola

    2016-04-01

    Multiple myeloma (MM) is characterized by severely imbalanced bone remodeling. In this study, we investigated the potential effect of proteasome inhibitors (PIs), a class of drugs known to stimulate bone formation, on the mechanisms involved in osteocyte death induced by MM cells. First, we performed a histological analysis of osteocyte viability on bone biopsies on a cohort of 37 MM patients with symptomatic disease. A significantly higher number of viable osteocytes was detected in patients treated with a bortezomib (BOR)-based regimen compared with those treated without BOR. Interestingly, both osteocyte autophagy and apoptosis were affected in vivo by BOR treatment. Thereafter, we checked the in vitro effect of BOR to understand the mechanisms whereby BOR maintains osteocyte viability in bone from MM patients. We found that osteocyte and preosteocyte autophagic death was triggered during coculturing with MM cells. Our evaluation was conducted by analyzing either autophagy markers microtubule-associated protein light chain 3 beta (LC3B) and SQSTM1/sequestome 1 (p62) levels, or the cell ultrastructure by transmission electron microscopy. PIs were found to increase the basal levels of LC3 expression in the osteocytes while blunting the myeloma-induced osteocyte death. PIs also reduced the autophagic death of osteocytes induced by high-dose dexamethasone (DEX) and potentiated the anabolic effect of PTH(1-34). Our data identify osteocyte autophagy as a new potential target in MM bone disease and support the use of PIs to maintain osteocyte viability and improve bone integrity in MM patients. PMID:26551485

  16. Induction of cell death by the novel proteasome inhibitor marizomib in glioblastoma in vitro and in vivo.

    PubMed

    Manton, Christa A; Johnson, Blake; Singh, Melissa; Bailey, Cavan P; Bouchier-Hayes, Lisa; Chandra, Joya

    2016-01-01

    New therapies for glioblastoma (GBM) are needed, as five-year survival is <10%. The proteasome inhibitor marizomib (MRZ) has inhibitory and death-inducing properties unique from previous inhibitors such as bortezomib (BTZ), and has not been well examined in GBM. We evaluated the mechanism of death and in vivo properties of MRZ in GBM. The activation kinetics of initiator caspases 2, 8, and 9 were assessed using chemical and knockdown strategies to determine their contribution to cell death. Blood brain barrier permeance and proteasome inhibition by MRZ and BTZ were examined in an orthotopic GBM model. Blockade of caspase 9, relative to other caspases, was most protective against both MRZ and BTZ. Only MRZ increased the proteasome substrate p27 in orthotopic brain tumors after a single injection, while both MRZ and BTZ increased p21 levels after multiple treatments. Cleavage of caspase substrate lamin A was increased in orthotopic brain tumors from mice treated with MRZ or BTZ and the histone deacetylase inhibitor vorinostat. Our data indicate that MRZ induces caspase 9-dependent death in GBM, suggesting drug efficacy biomarkers and possible resistance mechanisms. MRZ reaches orthotopic brain tumors where it inhibits proteasome function and increases death in combination with vorinostat. PMID:26804704

  17. Clioquinol and pyrrolidine dithiocarbamate complex with copper to form proteasome inhibitors and apoptosis inducers in human breast cancer cells

    PubMed Central

    Daniel, Kenyon G; Chen, Di; Orlu, Shirley; Cui, Qiuzhi Cindy; Miller, Fred R; Dou, Q Ping

    2005-01-01

    Introduction A physiological feature of many tumor tissues and cells is the tendency to accumulate high concentrations of copper. While the precise role of copper in tumors is cryptic, copper, but not other trace metals, is required for angiogenesis. We have recently reported that organic copper-containing compounds, including 8-hydroxyquinoline-copper(II) and 5,7-dichloro-8-hydroxyquinoline-copper(II), comprise a novel class of proteasome inhibitors and tumor cell apoptosis inducers. In the current study, we investigate whether clioquinol (CQ), an analog of 8-hydroxyquinoline and an Alzheimer's disease drug, and pyrrolidine dithiocarbamate (PDTC), a known copper-binding compound and antioxidant, can interact with copper to form cancer-specific proteasome inhibitors and apoptosis inducers in human breast cancer cells. Tetrathiomolybdate (TM), a strong copper chelator currently being tested in clinical trials, is used as a comparison. Methods Breast cell lines, normal, immortalized MCF-10A, premalignant MCF10AT1K.cl2, and malignant MCF10DCIS.com and MDA-MB-231, were treated with CQ or PDTC with or without prior interaction with copper, followed by measurement of proteasome inhibition and cell death. Inhibition of the proteasome was determined by levels of the proteasomal chymotrypsin-like activity and ubiquitinated proteins in protein extracts of the treated cells. Apoptotic cell death was measured by morphological changes, Hoechst staining, and poly(ADP-ribose) polymerase cleavage. Results When in complex with copper, both CQ and PDTC, but not TM, can inhibit the proteasome chymotrypsin-like activity, block proliferation, and induce apoptotic cell death preferentially in breast cancer cells, less in premalignant breast cells, but are non-toxic to normal/non-transformed breast cells at the concentrations tested. In contrast, CQ, PDTC, TM or copper alone had no effects on any of the cells. Breast premalignant or cancer cells that contain copper at concentrations

  18. NMDAR-dependent proteasome activity in the gustatory cortex is necessary for conditioned taste aversion.

    PubMed

    Rosenberg, Tali; Elkobi, Alina; Dieterich, Daniela C; Rosenblum, Kobi

    2016-04-01

    Taste information is processed in different brain structures in the mammalian brain, including the gustatory cortex (GC), which resides within the insular cortex. N-methyl-d-aspartate receptor (NMDAR) activity in the GC is necessary for the acquisition of conditioned taste aversion (CTA) but not positive novel taste learning. Previous studies have shown that taste memory consolidation requires intact protein synthesis in the GC. In addition, the direct involvement of translation initiation and elongation factors was documented in the GC during taste learning. However, protein expression is defined by protein synthesis, degradation, and localization. Protein degradation is critical for the consolidation and reconsolidation of other forms of learning, such as fear learning and addiction behavior, but its role in cortical-dependent learning is not clear. Here, we show for the first time that proteasome activity is specifically increased in the GC 4h following experiencing of a novel taste. This increase in proteasome activity was abolished by local administration to the GC of the NMDA antagonist, APV, as well as a CaMKII inhibitor, at the time of acquisition. In addition, local application of lactacystin, a proteasome inhibitor, resulted in impaired CTA, but not novel taste learning. These results suggest that NMDAR-dependent proteasome activity in the GC participates in the association process between novel taste experience and negative visceral sensation. PMID:26785229

  19. Tyrosine Hydroxylase Is Short-Term Regulated by the Ubiquitin-Proteasome System in PC12 Cells and Hypothalamic and Brainstem Neurons from Spontaneously Hypertensive Rats: Possible Implications in Hypertension

    PubMed Central

    Carbajosa, Nadia A. Longo; Corradi, Gerardo; Verrilli, María A. Lopez; Guil, María J.; Vatta, Marcelo S.; Gironacci, Mariela M.

    2015-01-01

    Aberrations in the ubiquitin-proteasome system (UPS) are implicated in the pathogenesis of various diseases. Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamines biosynthesis, is involved in hypertension development. In this study we investigated whether UPS regulated TH turnover in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats (SHR) and whether this system was impaired in hypertension. PC12 cells were exposed to proteasome or lysosome inhibitors and TH protein level evaluated by Western blot. Lactacystin, a proteasome inhibitor, induced an increase of 86±15% in TH levels after 30 min of incubation, then it started to decrease up to 6 h to reach control levels and finally it rose up to 35.2±8.5% after 24 h. Bafilomycin, a lysosome inhibitor, did not alter TH protein levels during short times, but it increased TH by 92±22% above basal after 6 h treatment. Before degradation proteasome substrates are labeled by conjugation with ubiquitin. Efficacy of proteasome inhibition on TH turnover was evidenced by accumulation of ubiquitinylated TH after 30 min. Further, the inhibition of proteasome increased the quantity of TH phosphorylated at Ser40, which is essential for TH activity, by 2.7±0.3 fold above basal. TH protein level was upregulated in neurons from hypothalami and brainstem of SHR when the proteasome was inhibited during 30 min, supporting that neuronal TH is also short-term regulated by the proteasome. Since the increased TH levels reported in hypertension may result from proteasome dysfunction, we evaluate proteasme activity. Proteasome activity was significantly reduced by 67±4% in hypothalamic and brainstem neurons from SHR while its protein levels did not change. Present findings show that TH is regulated by the UPS. The impairment in proteasome activity observed in SHR neurons may be one of the causes of the increased TH protein levels reported in hypertension. PMID:25710381

  20. Shikonin, dually functions as a proteasome inhibitor and a necroptosis inducer in multiple myeloma cells.

    PubMed

    Wada, Naoko; Kawano, Yawara; Fujiwara, Shiho; Kikukawa, Yoshitaka; Okuno, Yutaka; Tasaki, Masayoshi; Ueda, Mitsuharu; Ando, Yukio; Yoshinaga, Kazuya; Ri, Masaki; Iida, Shinsuke; Nakashima, Takayuki; Shiotsu, Yukimasa; Mitsuya, Hiroaki; Hata, Hiroyuki

    2015-03-01

    Shikonin (SHK), a natural small agent (MW 288.3), reportedly induces cell death in various tumor cells. We have found that SHK also exerts potent cytocidal effects on human multiple myeloma (MM) cells, but its anticancer mechanism in MM cells remains to be elucidated. SHK at 2.5-5 µM induced apoptosis in seven MM cell lines, including the bortezomib-resistant cell line KMS11/BTZ. The IC50 value of SHK against KMS11/BTZ was comparable to that of a parental cell line KMS11 (1.1 and 1.56 µM, respectively). SHK induces accumulation of ubiquitinated proteins and activates XBP-1 in MM cells, suggesting that SHK functions as a proteasome inhibitor, eventually inducing ER stress-associated apoptosis. SHK increases levels of HSP70/72, which protects cells from apoptosis, and exerts greater cytocidal effects in combination with the HSP70/72 inhibitor VER-155008. At higher concentrations (10-20 µM), SHK induced cell death, which was completely inhibited by a necroptosis inhibitor, necrostatin-1 (Nec-1), while the cytocidal activity was unaffected by Z-VAD-FMK, strongly suggesting that cell death is induced by SHK at high concentrations through necroptosis. The present data show for the first time that SHK induces cell death in MM cells. SHK efficiently induces apoptosis and combination of heat shock protein inhibitor with low dose SHK enhances apoptosis, while high dose SHK induces necroptosis in MM cells. These findings together support the use of SHK as a potential therapeutic agent for MM. PMID:25530098

  1. Marchantin M: a novel inhibitor of proteasome induces autophagic cell death in prostate cancer cells

    PubMed Central

    Jiang, H; Sun, J; Xu, Q; Liu, Y; Wei, J; Young, C Y F; Yuan, H; Lou, H

    2013-01-01

    We previously reported that marchantin M (Mar) is an active agent to induce apoptosis in human prostate cancer (PCa), but the molecular mechanisms of action remain largely unknown. Here, we demonstrate that Mar potently inhibited chymotrypsin-like and peptidyl-glutamyl peptide-hydrolyzing activities of 20S proteasome both in in vitro and intracellular systems and significantly induced the accumulation of polyubiquitinated proteins in PCa cells. The computational modeling analysis suggested that Mar non-covalently bound to active sites of proteasome β5 and β1 subunits, resulting in a non-competitive inhibition. Proteasome inhibition by Mar subsequently resulted in endoplasmic reticulum (ER) stress, as evidenced by elevated glucose-regulated protein 78 and CHOP, increased phospho-eukaryotic translation initiation factor 2α (eIF2α), splicing of X-box-binding protein-1 and dilation of the ER. However, Mar-mediated cell death was not completely impaired by a pan inhibitor of caspases. Further studies revealed that the Mar-induced cell death was greatly associated with the activation of autophagy, as indicated by the significant induction of microtubule-associated protein-1 light chain-3 beta (LC3B) expression and conversion. Electron microscopic and green fluorescent protein-tagged LC3B analyses further demonstrated the ability of autophagy induction by Mar. Time kinetic studies revealed that Mar induced a rapid and highly sustained processing of LC3B in treated cells and simultaneously decreased the expression of p62/SQSTM1. Pharmacological blockade or knockdown of LC3B and Atg5 attenuated Mar-mediated cell death. The autophagic response triggered by Mar required the activation of RNA-dependent protein kinase-like ER kinase/eIF2α and suppression of the phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin axis via preventing activation and expression of Akt. Our results identified a novel mechanism for the cytotoxic effect of Mar, which strengthens it as

  2. Proteasome inhibitors act as bifunctional antagonists of human immunodeficiency virus type 1 latency and replication

    PubMed Central

    2013-01-01

    Background Existing highly active antiretroviral therapy (HAART) effectively controls viral replication in human immunodeficiency virus type 1 (HIV-1) infected individuals but cannot completely eradicate the infection, at least in part due to the persistence of latently infected cells. One strategy that is being actively pursued to eliminate the latent aspect of HIV-1 infection involves therapies combining latency antagonists with HAART. However, discordant pharmacokinetics between these types of drugs can potentially create sites of active viral replication within certain tissues that might be impervious to HAART. Results A preliminary reverse genetic screen indicated that the proteasome might be involved in the maintenance of the latent state. This prompted testing to determine the effects of proteasome inhibitors (PIs) on latently infected cells. Experiments demonstrated that PIs effectively activated latent HIV-1 in several model systems, including primary T cell models, thereby defining PIs as a new class of HIV-1 latency antagonists. Expanding upon experiments from previous reports, it was also confirmed that PIs inhibit viral replication. Moreover, it was possible to show that PIs act as bifunctional antagonists of HIV-1. The data indicate that PIs activate latent provirus and subsequently decrease viral titers and promote the production of defective virions from activated cells. Conclusions These results represent a proof-of-concept that bifunctional antagonists of HIV-1 can be developed and have the capacity to ensure precise tissue overlap of anti-latency and anti-replication functions, which is of significant importance in the consideration of future drug therapies aimed at viral clearance. PMID:24156270

  3. Detrimental Effect of the Proteasome Inhibitor, Bortezomib in Bacterial Superantigen- and Lipopolysaccharide-induced Systemic Inflammation

    PubMed Central

    Tilahun, Ashenafi Y; Theuer, Jayne E; Patel, Robin; David, Chella S; Rajagopalan, Govindarajan

    2010-01-01

    Bacterial superantigen (BSAg)–induced toxic shock syndrome (TSS) and bacterial lipopolysaccharide (LPS)–induced shock are characterized by severe systemic inflammation. As nuclear factor κB (NFκB) plays an important role in inflammation and bortezomib, a proteasome inhibitor widely used in cancer chemotherapy, is a potent inhibitor of NFκB activation, we evaluated the therapeutic and prophylactic use of bortezomib in these conditions using murine models. Bortezomib prophylaxis significantly reduced serum levels of many cytokines and chemokines induced by BSAg. However, at 3 hours, serum level of TNF-a, an important cytokine implicated in TSS, was significantly reduced but not abolished. At 6 hours, there was no difference in the serum TNF-a levels between bortezomib treated and untreated mice challenged with staphylococcal enterotoxin B (SEB). Paradoxically, all mice treated with bortezomib either before or after BSAg challenge succumbed to TSS. Neither bortezomib nor BSAg was lethal if given alone. Serum biochemical parameters and histopathological findings suggested acute liver failure as the possible cause of mortality. Liver tissue from SEB-challenged mice treated with bortezomib showed a significant reduction in NFκB activation. Because NFκB-dependent antiapoptotic pathways protect hepatocytes from TNF-α-induced cell death, inhibition of NFκB brought forth by bortezomib in the face of elevated TNF-α levels caused by BSAg or LPS is detrimental. PMID:20372109

  4. Detrimental effect of the proteasome inhibitor, bortezomib in bacterial superantigen- and lipopolysaccharide-induced systemic inflammation.

    PubMed

    Tilahun, Ashenafi Y; Theuer, Jayne E; Patel, Robin; David, Chella S; Rajagopalan, Govindarajan

    2010-06-01

    Bacterial superantigen (BSAg)-induced toxic shock syndrome (TSS) and bacterial lipopolysaccharide (LPS)-induced shock are characterized by severe systemic inflammation. As nuclear factor kappaB (NF kappaB) plays an important role in inflammation and bortezomib, a proteasome inhibitor widely used in cancer chemotherapy, is a potent inhibitor of NF kappaB activation, we evaluated the therapeutic and prophylactic use of bortezomib in these conditions using murine models. Bortezomib prophylaxis significantly reduced serum levels of many cytokines and chemokines induced by BSAg. However, at 3 hours, serum level of TNF-a, an important cytokine implicated in TSS, was significantly reduced but not abolished. At 6 hours, there was no difference in the serum TNF-a levels between bortezomib treated and untreated mice challenged with staphylococcal enterotoxin B (SEB). Paradoxically, all mice treated with bortezomib either before or after BSAg challenge succumbed to TSS. Neither bortezomib nor BSAg was lethal if given alone. Serum biochemical parameters and histopathological findings suggested acute liver failure as the possible cause of mortality. Liver tissue from SEB-challenged mice treated with bortezomib showed a significant reduction in NF kappaB activation. Because NF kappaB-dependent antiapoptotic pathways protect hepatocytes from TNF-alpha-induced cell death, inhibition of NF kappaB brought forth by bortezomib in the face of elevated TNF-alpha levels caused by BSAg or LPS is detrimental. PMID:20372109

  5. Synergistic anti-myeloma activity of the proteasome inhibitor marizomib and the IMiD immunomodulatory drug pomalidomide.

    PubMed

    Das, Deepika S; Ray, Arghya; Song, Yan; Richardson, Paul; Trikha, Mohit; Chauhan, Dharminder; Anderson, Kenneth C

    2015-12-01

    The proteasome inhibitor bortezomib is an effective therapy for the treatment of relapsed and refractory multiple myeloma (RRMM); however, prolonged treatment can be associated with toxicity, peripheral neuropathy and drug resistance. Our earlier studies showed that the novel proteasome inhibitor marizomib is distinct from bortezomib in its chemical structure, mechanisms of action and effects on proteasomal activities, and that it can overcome bortezomib resistance. Pomalidomide, like lenalidomide, has potent immunomodulatory activity and has been approved by the US Food and Drug Administration for the treatment of RRMM. Here, we demonstrate that combining low concentrations of marizomib with pomalidomide induces synergistic anti-MM activity. Marizomib plus pomalidomide-induced apoptosis is associated with: (i) activation of caspase-8, caspase-9, caspase-3 and PARP cleavage, (ii) downregulation of cereblon (CRBN), IRF4, MYC and MCL1, and (iii) suppression of chymotrypsin-like, caspase-like, and trypsin-like proteasome activities. CRBN-siRNA attenuates marizomib plus pomalidomide-induced MM cells death. Furthermore, marizomib plus pomalidomide inhibits the migration of MM cells and tumour-associated angiogenesis, as well as overcomes cytoprotective effects of bone marrow microenvironment. In human MM xenograft model studies, the combination of marizomib and pomalidomide is well tolerated, inhibits tumour growth and prolongs survival. These preclinical studies provide the rationale for on-going clinical trials of combined marizomib and pomalidomide to improve outcome in patients with RRMM. PMID:26456076

  6. Antileukemic Activity and Mechanism of Drug Resistance to the Marine Salinispora tropica Proteasome Inhibitor Salinosporamide A (Marizomib)

    PubMed Central

    Niewerth, Denise; Jansen, Gerrit; Riethoff, Lesley F. V.; van Meerloo, Johan; Kale, Andrew J.; Moore, Bradley S.; Assaraf, Yehuda G.; Anderl, Janet L.; Zweegman, Sonja; Kaspers, Gertjan J. L.

    2014-01-01

    Salinosporamide A (NPI-0052, marizomib) is a naturally occurring proteasome inhibitor derived from the marine actinobacterium Salinispora tropica, and represents a promising clinical agent in the treatment of hematologic malignancies. Recently, these actinobacteria were shown to harbor self-resistance properties to salinosporamide A by expressing redundant catalytically active mutants of the 20S proteasome β-subunit, reminiscent of PSMB5 mutations identified in cancer cells with acquired resistance to the founding proteasome inhibitor bortezomib (BTZ). Here, we assessed the growth inhibitory potential of salinosporamide A in human acute lymphocytic leukemia CCRF-CEM cells, and its 10-fold (CEM/BTZ7) and 123-fold (CEM/BTZ200) bortezomib-resistant sublines harboring PSMB5 mutations. Parental cells displayed sensitivity to salinosporamide A (IC50 = 5.1 nM), whereas their bortezomib-resistant sublines were 9- and 17-fold cross-resistant to salinosporamide A, respectively. Notably, combination experiments of salinosporamide A and bortezomib showed synergistic activity in CEM/BTZ200 cells. CEM cells gradually exposed to 20 nM salinosporamide A (CEM/S20) displayed stable 5-fold acquired resistance to salinosporamide A and were 3-fold cross-resistant to bortezomib. Consistent with the acquisition of a PSMB5 point mutation (M45V) in CEM/S20 cells, salinosporamide A displayed a markedly impaired capacity to inhibit β5-associated catalytic activity. Last, compared with parental CEM cells, CEM/S20 cells exhibited up to 2.5-fold upregulation of constitutive proteasome subunits, while retaining unaltered immunoproteasome subunit expression. In conclusion, salinosporamide A displayed potent antileukemic activity against bortezomib-resistant leukemia cells. β-Subunit point mutations as a common feature of acquired resistance to salinosporamide A and bortezomib in hematologic cells and S. tropica suggest an evolutionarily conserved mechanism of resistance to proteasome

  7. Crystal structure of N-{N-[N-acetyl-(S)-leucyl]-(S)-leucyl}norleucinal (ALLN), an inhibitor of proteasome

    DOE PAGESBeta

    Czerwinski, Andrzej; Basava, Channa; Dauter, Miroslawa; Dauter, Zbigniew

    2015-03-01

    The title compound, C20H37N3O4, also known by the acronym ALLN, is a tripeptidic inhibitor of the proteolytic activity of the proteasomes, enzyme complexes implicated in several neurodegenerative diseases and other disorders, including cancer. Thus, the crystal structure of ALLN, solved from synchrotron radiation diffraction data, revealed the molecules in extended conformation of the backbone and engaging all peptide N and O atoms in intermolecular hydrogen bonds forming an infinite antiparallel β-sheet.

  8. E11/Podoplanin Protein Stabilization Through Inhibition of the Proteasome Promotes Osteocyte Differentiation in Murine in Vitro Models.

    PubMed

    Staines, Katherine A; Prideaux, Matt; Allen, Steve; Buttle, David J; Pitsillides, Andrew A; Farquharson, Colin

    2016-06-01

    The transmembrane glycoprotein E11 is considered critical in early osteoblast-osteocyte transitions (osteocytogenesis), however its function and regulatory mechanisms are still unknown. Using the late osteoblast MLO-A5 cell line we reveal increased E11 protein/mRNA expression (P < 0.001) concomitant with extensive osteocyte dendrite formation and matrix mineralization (P < 0.001). Transfection with E11 significantly increased mRNA levels (P < 0.001), but immunoblotting failed to detect any correlative increases in E11 protein levels, suggestive of post-translational degradation. We found that exogenous treatment of MLO-A5 and osteocytic IDG-SW3 cells with 10 μM ALLN (calpain and proteasome inhibitor) stabilized E11 protein levels and induced a profound increase in osteocytic dendrite formation (P < 0.001). Treatment with other calpain inhibitors failed to promote similar osteocytogenic changes, suggesting that these effects of ALLN rely upon its proteasome inhibitor actions. Accordingly we found that proteasome-selective inhibitors (MG132/lactacystin/ Bortezomib/Withaferin-A) produced similar dose-dependent increases in E11 protein levels in MLO-A5 and primary osteoblast cells. This proteasomal targeting was confirmed by immunoprecipitation of ubiquitinylated proteins, which included E11, and by increased levels of ubiquitinylated E11 protein upon addition of the proteasome inhibitors MG132/Bortezomib. Activation of RhoA, the small GTPase, was found to be increased concomitant with the peak in E11 levels and its downstream signaling was also observed to promote MLO-A5 cell dendrite formation. Our data indicate that a mechanism reliant upon blockade of proteasome-mediated E11 destabilization contributes to osteocytogenesis and that this may involve downstream targeting of RhoA. This work adds to our mechanistic understanding of the factors regulating bone homeostasis, which may lead to future therapeutic approaches. PMID:26639105

  9. The effect of peptidic and non-peptidic proteasome inhibitors on the biological properties of Acanthamoeba castellanii belonging to the T4 genotype.

    PubMed

    Siddiqui, Ruqaiyyah; Saleem, Sahreena; Khan, Naveed Ahmed

    2016-09-01

    The treatment of Acanthamoeba infections remains problematic, suggesting that new targets and/or chemotherapeutic agents are needed. Bioassay-guided screening of drugs that are clinically-approved for non-communicable diseases against opportunistic eukaryotic pathogens is a viable strategy. With known targets and mode of action, such drugs can advance to clinical trials at a faster pace. Recently Bortezomib (proteasome inhibitor) has been approved by FDA in the treatment of multiple myeloma. As proteasomal pathways are well known regulators of a variety of eukaryotic cellular functions, the overall aim of the present study was to study the effects of peptidic and non-peptidic proteasome inhibitors on the biology and pathogenesis of Acanthamoeba castellanii of the T4 genotype, in vitro. Zymographic assays revealed that inhibition of proteasome had detrimental effects on the extracellular proteolytic activities of A. castellanii. Proteasome inhibition affected A. castellanii growth (using amoebistatic assays), but not viability of A. castellanii. Importantly, proteasome inhibitors affected encystation as determined by trophozoite transformation into the cyst form, as well as excystation, as determined by cyst transformation into the trophozoite form. The ability of proteasome inhibitor to block Acanthamoeba differentiation is significant, as it presents a major challenge in the successful treatment of Acanthamoeba infection. As these drugs are used clinically against non-communicable diseases, the findings reported here have the potential to be tested in a clinical setting against amoebic infections. PMID:27327524

  10. Selective overproduction of the proteasome inhibitor salinosporamide A via precursor pathway regulation

    PubMed Central

    Lechner, Anna; Eustáquio, Alessandra S.; Gulder, Tobias A. M.; Hafner, Mathias; Moore, Bradley S.

    2011-01-01

    The chlorinated natural product salinosporamide A is a potent 20S proteasome inhibitor currently in clinical trials as an anticancer agent. To deepen our understanding of salinosporamide biosynthesis, we investigated the function of a LuxR-type pathway-specific regulatory gene, salR2, and observed a selective effect on the production of salinosporamide A over its less active aliphatic analogs. SalR2 was shown to specifically activate genes involved in the biosynthesis of the halogenated precursor chloroethylmalonyl-CoA, which is a dedicated precursor of salinosporamide A. Specifically, SalR2 activates transcription of two divergent operons – one of which contains the unique S-adenosyl-L-methionine-dependent chlorinase encoding gene salL. By applying this knowledge towards rational engineering, we were able to selectively double salinosporamide A production. This study exemplifies the specialized regulation of a polyketide precursor pathway and its application to the selective overproduction of a specific natural product congener. PMID:22195555

  11. Pulmonary Nocardiosis in a Multiple Myeloma Patient Treated with Proteasome Inhibitors

    PubMed Central

    Mendonca, Nikolai P.; Kadayakkara, Deepak K.; Forde, Inga C.; Rudkovaskaia, Anastasiia; Saul, Zane K.; Lobo, David J.

    2016-01-01

    Patient: Male, 71 Final Diagnosis: Pulmonary nocardiosis Symptoms: Cough • dyspnea • fever Medication: Carfillzomib Clinical Procedure: Bronchoscopy Specialty: Infectious Diseases Objective: Rare co-existance of disease or pathology Background: The use of proteasome inhibitors like Bortezomib to treat multiple myeloma has been associated with increased rates of opportunistic infections, including Nocardia, especially when lymphopenia is present. The prevalence or association of such infections with newer agents like Carfilzomib is not known. Case Report: A 71-year-old man with multiple myeloma presented with a 6-week history of respiratory symptoms and cyclic fevers. He was undergoing chemotherapy with Carfilzomib. Work-up revealed severe lymphopenia and a CT chest showed multiple lung nodules and a mass-like consolidation. He underwent a bronchoscopy, and respiratory cultures grew Nocardia species. He responded well to intravenous antibiotics with resolution of symptoms and CT findings. Conclusions: With the introduction of newer agents like Carfilzomib for the treatment of multiple myeloma, clinicians must maintain a high degree of suspicion for opportunistic infections to achieve early diagnosis and treatment. PMID:26861506

  12. Syntheses of C-13 and C-14-labeled versions of the investigational proteasome inhibitor MLN9708.

    PubMed

    Plesescu, Mihaela; Elliott, Eric L; Li, Yuexian; Prakash, Shimoga R

    2013-01-01

    MLN9708 (ixazomib citrate) is an investigational, orally bioavailable proteasome inhibitor that is under development by Millennium in clinical studies in both hematologic and nonhematologic malignancies. The stable isotope-labeled MLN9708 was required for bio-analytical studies. [(13) C9 ]-MLN9708 (11) was synthesized in seven steps from the uniformly labeled [(13) C6 ]-1,4-dichlorobenzene (3) and [1-(13) C]-acetyl chloride. Because of the presence of two chlorine atoms and a boron atom, compound 6 was further reacted with [(13) C2 ]-glycine to provide an internal standard that is well separated from the parent compound during mass spectrometric analysis. The radiolabeled version was prepared to support metabolite profiling and whole body autoradiography studies in experimental animals. [(14) C]-MLN9708 (19) was synthesized in six steps from commercially available [(14) C]-barium carbonate. The key intermediate, [carboxyl-(14) C]-2,5-dichlorobenzoic acid (14), was prepared by selective lithiation of 1-bromo-2,5-dichlorobenzene (12) followed by carbonation with [(14) C]-barium carbonate. In preparation for a one-time human absorption, distribution, metabolism and excretion (ADME) study, the stability of [(14) C]-MLN9708 and its precursors were also evaluated. PMID:24285522

  13. Metal-based 2,3-indolinedione derivatives as proteasome inhibitors and inducers of apoptosis in human cancer cells.

    PubMed

    Zhang, Pengfei; Bi, Caifeng; Schmitt, Sara M; Li, Xin; Fan, Yuhua; Zhang, Nan; Dou, Q Ping

    2014-09-01

    Proliferation and apoptotic pathways are tightly regulated in cells by the ubiquitin-proteasome system (UPS). Alterations in the UPS may result in cellular transformation or other pathological conditions. The proteasome is indeed often found to be overactive in cancer cells. It has been reported that 2,3-indolinedione (L), which exists in marine organisms, as well as in mammals, is a proteasome inhibitor. Studies have shown that metal-based complexes inhibit proteasome activity and induce apoptosis in certain human cancer cells. In the current study, we synthesized six novel metal-based complexes with derivatives of 2,3-indolinedione: [Cd (C15H11O3N2) (CH3COO)] (C1), [Cd (C15H11O2N2) (CH3COO)] (C2), [Co (C15H9O4N2) (CH3COO)] (C3), [Co (C15H11O2N2) (CH3COO)] (C4), [Zn (C19H14O3N3) (CH3COO)] (C5) and [Zn (C17H13O3N2) (CH3COO)] (C6). We sought to characterize and assess the proteasome inhibitory and anti-proliferative effects of these metal-based complexes in human breast (MDA-MB-231) and prostate (LNCaP and PC-3) cancer cells, in order to determine whether specific structures contribute to the inhibition of tumor proteasome activity and the induction of apoptosis. The results revealed that the complexes, C1, C3 and C5, but not their counterparts, C2, C4 and C6, inhibited the chymotrypsin-like activity of the human cancer cellular 26S proteasome; in addition, these complexes promoted the accumulation of the proteasome target protein, Bax, inhibited cell growth and induced apoptosis in a concentration- and time-dependent manner due to their unique structures. Our data suggest that the study of metal-based complexes, including aromatic ring structures with electron-attracting groups, may be an interesting research direction for the development of anticancer drugs. PMID:25017797

  14. Sorafenib enhances proteasome inhibitor-mediated cytotoxicity via inhibition of unfolded protein response and keratin phosphorylation

    SciTech Connect

    Honma, Yuichi; Harada, Masaru

    2013-08-15

    Hepatocellular carcinoma (HCC) is highly resistant to conventional systemic therapies and prognosis for advanced HCC patients remains poor. Recent studies of the molecular mechanisms responsible for tumor initiation and progression have identified several potential molecular targets in HCC. Sorafenib is a multi-kinase inhibitor shown to have survival benefits in advanced HCC. It acts by inhibiting the serine/threonine kinases and the receptor type tyrosine kinases. In preclinical experiments sorafenib had anti-proliferative activity in hepatoma cells and it reduced tumor angiogenesis and increased apoptosis. Here, we demonstrate for the first time that the cytotoxic mechanisms of sorafenib include its inhibitory effects on protein ubiquitination, unfolded protein response (UPR) and keratin phosphorylation in response to endoplasmic reticulum (ER) stress. Moreover, we show that combined treatment with sorafenib and proteasome inhibitors (PIs) synergistically induced a marked increase in cell death in hepatoma- and hepatocyte-derived cells. These observations may open the way to potentially interesting treatment combinations that may augment the effect of sorafenib, possibly including drugs that promote ER stress. Because sorafenib blocked the cellular defense mechanisms against hepatotoxic injury not only in hepatoma cells but also in hepatocyte-derived cells, we must be careful to avoid severe liver injury. -- Graphical abstract: Display Omitted -- Highlights: •We examined the cytotoxic mechanisms of sorafenib in hepatoma cells. •Sorafenib induces cell death via apoptotic and necrotic fashion. •Sorafenib inhibits protein ubiquitination and unfolded protein response. •Autophagy induced by sorafenib may affect its cytotoxicity. •Sorafenib inhibits keratin phosphorylation and cytoplasmic inclusion formation.

  15. A plastic SQSTM1/p62-dependent autophagic reserve maintains proteostasis and determines proteasome inhibitor susceptibility in multiple myeloma cells

    PubMed Central

    Milan, Enrico; Perini, Tommaso; Resnati, Massimo; Orfanelli, Ugo; Oliva, Laura; Raimondi, Andrea; Cascio, Paolo; Bachi, Angela; Marcatti, Magda; Ciceri, Fabio; Cenci, Simone

    2015-01-01

    Multiple myeloma (MM) is the paradigmatic proteasome inhibitor (PI) responsive cancer, but many patients fail to respond. An attractive target to enhance sensitivity is (macro)autophagy, recently found essential to bone marrow plasma cells, the normal counterpart of MM. Here, integrating proteomics with hypothesis-driven strategies, we identified the autophagic cargo receptor and adapter protein, SQSTM1/p62 as an essential component of an autophagic reserve that not only synergizes with the proteasome to maintain proteostasis, but also mediates a plastic adaptive response to PIs, and faithfully reports on inherent PI sensitivity. Lentiviral engineering revealed that SQSTM1 is essential for MM cell survival and affords specific PI protection. Under basal conditions, SQSTM1-dependent autophagy alleviates the degradative burden on the proteasome by constitutively disposing of substantial amounts of ubiquitinated proteins. Indeed, its inhibition or stimulation greatly sensitized to, or protected from, PI-induced protein aggregation and cell death. Moreover, under proteasome stress, myeloma cells selectively enhanced SQSTM1 de novo expression and reset its vast endogenous interactome, diverting SQSTM1 from signaling partners to maximize its association with ubiquitinated proteins. Saturation of such autophagic reserve, as indicated by intracellular accumulation of undigested SQSTM1-positive aggregates, specifically discriminated patient-derived myelomas inherently susceptible to PIs from primarily resistant ones. These aggregates correlated with accumulation of the endoplasmic reticulum, which comparative proteomics identified as the main cell compartment targeted by autophagy in MM. Altogether, the data integrate autophagy into our previously established proteasome load-versus-capacity model, and reveal SQSTM1 aggregation as a faithful marker of defective proteostasis, defining a novel prognostic and therapeutic framework for MM. PMID:26043024

  16. Identification of Potent and Selective Non-covalent Inhibitors of the Plasmodium falciparum Proteasome

    PubMed Central

    2015-01-01

    We have identified short N,C-capped peptides that selectively inhibit the proteasome of the malaria-causing pathogen Plasmodium falciparum. These compounds are highly potent in culture with no toxicity in host cells. One cyclic biphenyl ether compound inhibited intraerythrocytic growth of P. falciparum with an IC50 of 35 nM, and we show that even a pulse treatment with this cyclic peptide induced parasite death due to proteasome inhibition. These compounds represent promising new antimalarial agents that target the essential proteasomal machinery of the parasite without toxicity toward the host. PMID:25226494

  17. Selective intracellular delivery of proteasome inhibitors through pH-sensitive polymeric micelles directed to efficient antitumor therapy.

    PubMed

    Quader, S; Cabral, H; Mochida, Y; Ishii, T; Liu, X; Toh, K; Kinoh, H; Miura, Y; Nishiyama, N; Kataoka, K

    2014-08-28

    The ubiquitin-proteasome system is central in the regulation of cellular proteins controlling cell cycle progression and apoptosis, drawing much interest for developing effective targeted cancer therapies. Herein, we developed a novel pH-responsive polymeric-micelle-based carrier system to effectively deliver the proteasome inhibitor MG132 into cancer cells. MG132 is covalently bound to the block copolymer composed of polyethylene glycol (PEG) and polyaspartate through an acid-labile hydrazone bond. This bond is stable at physiological condition, but hydrolytically degradable in acidic compartments in the cell, such as late-endosomes and lysosomes, and thus, it was used for controlled release of MG132 after EPR-mediated preferential accumulation of the micelles into the tumor. MG132-loaded micelles have monodispersed size distribution with an average diameter of 45nm, and critical micelle concentration is well below 10(-7)M. In vitro studies against several cancer cell lines confirmed that MG132-loaded micelles retained the cytotoxic effect, and this activity was indeed due to the inhibition of proteasome by released MG132 from the micelles. Real-time in vitro confocal-microscopy experiments clearly indicated that MG132-conjugated micelles disintegrated only inside the target cells. By intravital confocal micro-videography, we also confirmed the prolonged circulation of MG132 loaded micelles in the bloodstream, which lead to tumor specific accumulation of micelles, as confirmed by in vivo imaging 24h after injection. These micelles showed significantly lower in vivo toxicity than free MG132, while achieving remarkable antitumor effect against a subcutaneous HeLa-luc tumor model. Our findings create a paradigm for future development of polymeric-micelle-based carrier system for other peptide aldehyde type proteasome inhibitors to make them effective cohort of the existing cancer therapeutic regiments. PMID:24892974

  18. Boronic acid-containing proteasome inhibitors: alert to potential pharmaceutical bioactivation.

    PubMed

    Li, Austin C; Yu, Erya; Ring, Steven C; Chovan, James P

    2013-04-15

    Medicinal chemists try to avoid certain organic functional groups, summarized in an ever-growing list, in order to avoid the potential bioactivation to reactive metabolites. To add to that alert list, we report herein that boronic acid-containing compound structures, such as those found in proteasome inhibitors bortezomib and ixazomib, can become bioactivated to chemically reactive imine amide metabolites. Test compounds, ixazomib and bortezomib, were incubated in vitro using human liver fractions containing cytosol and microsomes (S9) under conventional conditions in the presence of GSH. Metabolites were then analyzed using LC-MS(n) with or without online hydrogen-deuterium exchange (HDX) liquid chromatography coupled with an LTQ-Orbitrap. The exact mass measurements of both the precursor and product ions were acquired through data dependent acquisition and compared with theoretical values of proposed fragment ions. Upon deboronation catalyzed by cytochrome P450 enzymes, both test compounds formed imine amide metabolites that were identified by high resolution exact mass measurements in both normal aqueous and HDX HPLC-MS analysis. GSH conjugates were also identified and were postulated as nucleophilic addition of GSH to the imine amide metabolites. All mass spectrometric and HDX measurements of these GSH conjugates proved that the GSH unit was added to the carbon atom of the imine amide partial structure, hence demonstrating the electrophilic property of these imine amide metabolites. The awareness of the formation of electrophilic imine amide metabolites from boronic acid-containing compounds, where the boron atom is bonded to a carbon atom adjacent to an amide nitrogen, should help in drug candidate design and optimization with regard to avoiding potential bioactivation. PMID:23514361

  19. Catabolism of endogenous and overexpressed APH1a and PEN2: evidence for artifactual involvement of the proteasome in the degradation of overexpressed proteins

    PubMed Central

    Dunys, Julie; Kawarai, Toshitaka; Wilk, Sherwin; St. George-Hyslop, Peter; Alves Da Costa, Cristine; Checler, Frédéric

    2005-01-01

    PS (presenilin)-dependent γ-secretase occurs as a high-molecular-mass complex composed of either PS1 or PS2 associated with Nct (nicastrin), PEN2 (presenilin enhancer 2 homologue) and APH1 (anterior pharynx defective 1 homologue). Numerous reports have documented the very complicated physical and functional cross-talk between these proteins that ultimately governs the biological activity of the γ-secretase, but very few studies examined the fate of the components of the complex. We show that, in both HEK-293 cells and the TSM1 neuronal cell line, the immunoreactivities of overexpressed myc-tagged-APH1a and -PEN2 were enhanced by the proteasome inhibitors ZIE and lactacystin, whereas a broad range of protease inhibitors had no effect. By contrast, proteasome inhibitors were totally unable to affect the cellular expression of endogenous APH1aL and PEN2 in HEK-293 cells, TSM1 and primary cultured cortical neurons. To explain this apparent discrepancy, we examined the degradation of myc-tagged-APH1a and -PEN2, in vitro, by cell extracts containing endogenous proteasome and by purified 20S proteasome. Strikingly, myc-tagged-APH1a and -PEN2 resist proteolysis by endogenous proteasome and purified 20S proteasome. We also show that endogenous PEN2 expression was drastically higher in wild-type than in PS- and Nct-deficient fibroblasts and was enhanced by proteasome inhibitors only in the two deficient cell systems. However, here again, purified 20S proteasome appeared unable to cleave endogenous PEN2 present in PS-deficient fibroblasts. The levels of endogenous APH1aL-like immunoreactivity were not modified by proteasome inhibitors and were unaffected by PS deficiency. Altogether, our results indicate that endogenous PEN2 and APH1aL do not undergo proteasomal degradation under physiological conditions in HEK-293 cells, TSM1 cells and fibroblasts and that the clearance of PEN2 in PS- and Nct-deficient fibroblasts is not mediated by 20S proteasome. Whether the 26S

  20. Proteasome Inhibitors Block DNA Repair and Radiosensitize Non-Small Cell Lung Cancer

    PubMed Central

    Kushwaha, Deepa S.; Hsieh, Grace; Merzon, Dmitry; Rameseder, Jonathan; Chen, Clark C.; D’Andrea, Alan D.; Kozono, David

    2013-01-01

    Despite optimal radiation therapy (RT), chemotherapy and/or surgery, a majority of patients with locally advanced non-small cell lung cancer (NSCLC) fail treatment. To identify novel gene targets for improved tumor control, we performed whole genome RNAi screens to identify knockdowns that most reproducibly increase NSCLC cytotoxicity. These screens identified several proteasome subunits among top hits, including the topmost hit PSMA1, a component of the core 20 S proteasome. Radiation and proteasome inhibition showed synergistic effects. Proteasome inhibition resulted in an 80–90% decrease in homologous recombination (HR), a 50% decrease in expression of NF-κB-inducible HR genes BRCA1 and FANCD2, and a reduction of BRCA1, FANCD2 and RAD51 ionizing radiation-induced foci. IκBα RNAi knockdown rescued NSCLC radioresistance. Irradiation of mice with NCI-H460 xenografts after inducible PSMA1 shRNA knockdown markedly increased murine survival compared to either treatment alone. Proteasome inhibition is a promising strategy for NSCLC radiosensitization via inhibition of NF-κB-mediated expression of Fanconi Anemia/HR DNA repair genes. PMID:24040035

  1. Lipopolysaccharide Induces Degradation of Connexin43 in Rat Astrocytes via the Ubiquitin-Proteasome Proteolytic Pathway

    PubMed Central

    Liao, Chih-Kai; Jeng, Chung-Jiuan; Wang, Hwai-Shi; Wang, Shu-Huei; Wu, Jiahn-Chun

    2013-01-01

    The astrocytic syncytium plays a critical role in maintaining the homeostasis of the brain through the regulation of gap junction intercellular communication (GJIC). Changes to GJIC in response to inflammatory stimuli in astrocytes may have serious effects on the brain. We have previously shown that lipopolysaccharide (LPS) reduces connexin43 (Cx43) expression and GJIC in cultured rat astrocytes via a toll-like receptor 4-mediated signaling pathway. In the present study, treatment of astrocytes with LPS resulted in a significant increase in levels of the phosphorylated forms of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) -1, -2, and -3 for up to 18 h. An increase in nuclear transcription factor NF-κB levels was also observed after 8 h of LPS treatment and was sustained for up to 18 h. The LPS-induced decrease in Cx43 protein levels and inhibition of GJIC were blocked by the SAPK/JNK inhibitor SP600125, but not by the NF-κB inhibitor BAY11-7082. Following blockade of de novo protein synthesis by cycloheximide, LPS accelerated Cx43 degradation. Moreover, the LPS-induced downregulation of Cx43 was blocked following inhibition of 26S proteasome activity using the reversible proteasome inhibitor MG132 or the irreversible proteasome inhibitor lactacystin. Immunoprecipitation analyses revealed an increased association of Cx43 with both ubiquitin and E3 ubiquitin ligase Nedd4 in astrocytes after LPS stimulation for 6 h and this effect was prevented by SP600125. Taken together, these results suggest that LPS stimulation leads to downregulation of Cx43 expression and GJIC in rat astrocytes by activation of SAPK/JNK and the ubiquitin-proteasome proteolytic pathway. PMID:24236122

  2. A novel combination treatment for breast cancer cells involving BAPTA-AM and proteasome inhibitor bortezomib

    PubMed Central

    YERLIKAYA, AZMI; ERDOĞAN, ELIF; OKUR, EMRAH; YERLIKAYA, ŞERIFE; SAVRAN, BIRCAN

    2016-01-01

    Glucose-regulated protein 78 kDa/binding immunoglobulin protein (GRP78/BIP) is a well-known endoplasmic reticulum (ER) chaperone protein regulating ER stress by facilitating protein folding, assembly and Ca2+ binding. GRP78 is also a member of the heat shock protein 70 gene family and induces tumor cell survival and resistance to chemotherapeutics. Bortezomib is a highly specific 26S proteasome inhibitor that has been approved as treatment for patients with multiple myeloma. The present study first examined the dose- and time-dependent effects of bortezomib on GRP78 expression levels in the highly metastatic mouse breast cancer 4T1 cell line using western blot analysis. The analysis results revealed that GRP78 levels were significantly increased by bortezomib at a dose as low as 10 nM. Time-dependent experiments indicated that the accumulation of GRP78 was initiated after a 24 h incubation period following the addition of 10 nM bortezomib. Subsequently, the present study determined the half maximal inhibitory concentration of intracellular calcium chelator BAPTA-AM (13.6 µM) on 4T1 cells. The combination effect of BAPTA-AM and bortezomib on the 4T1 cells was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and WST-1 assays and an iCELLigence system. The results revealed that the combination of 10 nM bortezomib + 5 µM BAPTA-AM is more cytotoxic compared with monotherapies, including 10 nM bortezomib, 1 µM BAPTA-AM and 5 µM BAPTA-AM. In addition, the present results revealed that bortezomib + BAPTA-AM combination causes cell death through the induction of apoptosis. The present results also revealed that bortezomib + BAPTA-AM combination-induced apoptosis is associated with a clear increase in the phosphorylation of stress-activated protein kinase/Jun amino-terminal kinase SAPK/JNK. Overall, the present results suggest that bortezomib and BAPTA-AM combination therapy may be a novel therapeutic strategy for breast cancer treatment

  3. A novel orally active proteasome inhibitor ONX 0912 triggers in vitro and in vivo cytotoxicity in multiple myeloma.

    PubMed

    Chauhan, Dharminder; Singh, Ajita V; Aujay, Monette; Kirk, Christopher J; Bandi, Madhavi; Ciccarelli, Bryan; Raje, Noopur; Richardson, Paul; Anderson, Kenneth C

    2010-12-01

    Bortezomib therapy has proven successful for the treatment of relapsed, relapsed/refractory, and newly diagnosed multiple myeloma (MM). At present, bortezomib is available as an intravenous injection, and its prolonged treatment is associated with toxicity and development of drug resistance. Here we show that the novel proteasome inhibitor ONX 0912, a tripeptide epoxyketone, inhibits growth and induces apoptosis in MM cells resistant to conventional and bortezomib therapies. The anti-MM activity of ONX-0912 is associated with activation of caspase-8, caspase-9, caspase-3, and poly(ADP) ribose polymerase, as well as inhibition of migration of MM cells and angiogenesis. ONX 0912, like bortezomib, predominantly inhibits chymotrypsin-like activity of the proteasome and is distinct from bortezomib in its chemical structure. Importantly, ONX 0912 is orally bioactive. In animal tumor model studies, ONX 0912 significantly reduced tumor progression and prolonged survival. Immununostaining of MM tumors from ONX 0912-treated mice showed growth inhibition, apoptosis, and a decrease in associated angiogenesis. Finally, ONX 0912 enhances anti-MM activity of bortezomib, lenalidomide dexamethasone, or pan-histone deacetylase inhibitor. Taken together, our study provides the rationale for clinical protocols evaluating ONX 0912, either alone or in combination, to improve patient outcome in MM. PMID:20805366

  4. Combination of novel proteasome inhibitor NPI-0052 and lenalidomide trigger in vitro and in vivo synergistic cytotoxicity in multiple myeloma.

    PubMed

    Chauhan, Dharminder; Singh, Ajita V; Ciccarelli, Bryan; Richardson, Paul G; Palladino, Michael A; Anderson, Kenneth C

    2010-01-28

    Our recent study demonstrated that a novel proteasome inhibitor NPI-0052 is distinct from bortezomib (Velcade) and, importantly, triggers apoptosis in multiple myeloma (MM) cells resistant to bortezomib. Here we demonstrate that combining NPI-0052 and lenalidomide (Revlimid) induces synergistic anti-MM activity in vitro using MM-cell lines or patient MM cells. NPI-0052 plus lenalidomide-induced apoptosis is associated with (1) activation of caspase-8, caspase-9, caspase-12, caspase-3, and poly(ADP) ribose polymerase; (2) activation of BH-3 protein BIM; (3) translocation of BIM to endoplasmic reticulum; (4) inhibition of migration of MM cells and angiogenesis; and (5) suppression of chymotrypsin-like, caspase-like, and trypsin-like proteasome activities. Importantly, blockade of BIM using siRNA significantly abrogates NPI-0052 plus lenalidomide-induced apoptosis. Furthermore, studies using biochemical inhibitors of caspase-8 versus caspase-9 demonstrate that NPI-0052 plus lenalidomide-triggered apoptosis is primarily dependent on caspase-8 signaling. In animal tumor model studies, low-dose combination of NPI-0052 and lenalidomide is well tolerated, significantly inhibits tumor growth, and prolongs survival. Taken together, our study provides the preclinical rationale for clinical protocols evaluating lenalidomide together with NPI-0052 to improve patient outcome in MM. PMID:19965674

  5. Characterization of a new series of non-covalent proteasome inhibitors with exquisite potency and selectivity for the 20S β5-subunit

    PubMed Central

    Blackburn, Christopher; Gigstad, Kenneth M.; Hales, Paul; Garcia, Khristofer; Jones, Matthew; Bruzzese, Frank J.; Barrett, Cynthia; Liu, Jane X.; Soucy, Teresa A.; Sappal, Darshan S.; Bump, Nancy; Olhava, Edward J.; Fleming, Paul; Dick, Lawrence R.; Tsu, Christopher; Sintchak, Michael D.; Blank, Jonathan L.

    2010-01-01

    The mammalian 26S proteasome is a 2500 kDa multi-catalytic complex involved in intracellular protein degradation. We describe the synthesis and properties of a novel series of non-covalent di-peptide inhibitors of the proteasome used on a capped tri-peptide that was first identified by high-throughput screening of a library of approx. 350000 compounds for inhibitors of the ubiquitin–proteasome system in cells. We show that these compounds are entirely selective for the β5 (chymotrypsin-like) site over the β1 (caspase-like) and β2 (trypsin-like) sites of the 20S core particle of the proteasome, and over a panel of less closely related proteases. Compound optimization, guided by X-ray crystallography of the liganded 20S core particle, confirmed their non-covalent binding mode and provided a structural basis for their enhanced in vitro and cellular potencies. We demonstrate that such compounds show low nanomolar IC50 values for the human 20S β5 site in vitro, and that pharmacological inhibition of this site in cells is sufficient to potently inhibit the degradation of a tetra-ubiquitin–luciferase reporter, activation of NFκB (nuclear factor κB) in response to TNF-α (tumour necrosis factor-α) and the proliferation of cancer cells. Finally, we identified capped di-peptides that show differential selectivity for the β5 site of the constitutively expressed proteasome and immunoproteasome in vitro and in B-cell lymphomas. Collectively, these studies describe the synthesis, activity and binding mode of a new series of non-covalent proteasome inhibitors with unprecedented potency and selectivity for the β5 site, and which can discriminate between the constitutive proteasome and immunoproteasome in vitro and in cells. PMID:20632995

  6. Characterization of a new series of non-covalent proteasome inhibitors with exquisite potency and selectivity for the 20S [beta]5-subunit

    SciTech Connect

    Blackburn, Christopher; Gigstad, Kenneth M.; Hales, Paul; Garcia, Khristofer; Jones, Matthew; Bruzzese, Frank J.; Barrett, Cynthia; Liu, Jane X.; Soucy, Teresa A.; Sappal, Darshan S.; Bump, Nancy; Olhava, Edward J.; Fleming, Paul; Dick, Lawrence R.; Tsu, Christopher; Sintchak, Michael D.; Blank, Jonathan L.

    2012-04-30

    The mammalian 26S proteasome is a 2500 kDa multi-catalytic complex involved in intracellular protein degradation. We describe the synthesis and properties of a novel series of non-covalent di-peptide inhibitors of the proteasome used on a capped tri-peptide that was first identified by high-throughput screening of a library of approx. 350000 compounds for inhibitors of the ubiquitin-proteasome system in cells. We show that these compounds are entirely selective for the {beta}5 (chymotrypsin-like) site over the {beta}1 (caspase-like) and {beta}2 (trypsin-like) sites of the 20S core particle of the proteasome, and over a panel of less closely related proteases. Compound optimization, guided by X-ray crystallography of the liganded 20S core particle, confirmed their non-covalent binding mode and provided a structural basis for their enhanced in vitro and cellular potencies. We demonstrate that such compounds show low nanomolar IC{sub 50} values for the human 20S {beta}5 site in vitro, and that pharmacological inhibition of this site in cells is sufficient to potently inhibit the degradation of a tetra-ubiquitin-luciferase reporter, activation of NF{Kappa}B (nuclear factor {Kappa}B) in response to TNF-{alpha} (tumor necrosis factor-{alpha}) and the proliferation of cancer cells. Finally, we identified capped di-peptides that show differential selectivity for the {beta}5 site of the constitutively expressed proteasome and immunoproteasome in vitro and in B-cell lymphomas. Collectively, these studies describe the synthesis, activity and binding mode of a new series of non-covalent proteasome inhibitors with unprecedented potency and selectivity for the {beta}5 site, and which can discriminate between the constitutive proteasome and immunoproteasome in vitro and in cells.

  7. 3D-QSAR-aided design, synthesis, in vitro and in vivo evaluation of dipeptidyl boronic acid proteasome inhibitors and mechanism studies.

    PubMed

    Lei, Meng; Feng, Huayun; Wang, Cheng; Li, Hailing; Shi, Jingmiao; Wang, Jia; Liu, Zhaogang; Chen, Shanshan; Hu, Shihe; Zhu, Yongqiang

    2016-06-01

    Proteasome had been clinically validated as an effective target for the treatment of cancers. Up to now, many structurally diverse proteasome inhibitors were discovered. And two of them were launched to treat multiple myeloma (MM) and mantle cell lymphoma (MCL). Based on our previous biological results of dipeptidyl boronic acid proteasome inhibitors, robust 3D-QSAR models were developed and structure-activity relationship (SAR) was summarized. Several structurally novel compounds were designed based on the theoretical models and finally synthesized. Biological results showed that compound 12e was as active as the standard bortezomib in enzymatic and cellular activities. In vivo pharmacokinetic profiles suggested compound 12e showed a long half-life, which indicated that it could be administered intravenously. Cell cycle analysis indicated that compound 12e inhibited cell cycle progression at the G2M stage. PMID:27117691

  8. The Proteasome Inhibitor, MG132, Attenuates Diabetic Nephropathy by Inhibiting SnoN Degradation In Vivo and In Vitro

    PubMed Central

    Huang, Wei; Yang, Chen; Nan, Qinling; Gao, Chenlin; Feng, Hong; Gou, Fang; Chen, Guo; Zhang, Zhihong; Yan, Pijun; Peng, Juan

    2014-01-01

    Transforming growth factor-β (TGF-β) has been shown to be involved in diabetic nephropathy (DN). The SnoN protein can regulate TGF-β signaling through interaction with Smad proteins. Recent studies have shown that SnoN is mainly degraded by the ubiquitin-proteasome pathway. However, the role of SnoN in the regulation of TGF-β/Smad signaling in DN is still unclear. In this study, diabetic rats were randomly divided into a diabetic control group (DC group) and a proteasome inhibitor (MG132) diabetes therapy group (DT group). Kidney damage parameters and the expression of SnoN, Smurf2, and TGF-β were observed. Simultaneously, we cultured rat glomerular mesangial cells (GMCs) stimulated with high glucose, and SnoN and Arkadia expression were measured. Results demonstrated that 24-hour urine protein, ACR, BUN, and the expression of Smurf2 and TGF-β were significantly increased (P < 0.05), whereas SnoN was significantly decreased in the DC group (P < 0.05). However, these changes diminished after treatment with MG132. SnoN expression in GMCs decreased significantly (P < 0.05), but Arkadia expression gradually increased due to high glucose stimulation (P < 0.05), which could be almost completely reversed by MG132 (P < 0.05). The present results support the hypothesis that MG132 may alleviate kidney damage by inhibiting SnoN degradation and TGF-β activation, suggesting that the ubiquitin-proteasome pathway may become a new therapeutic target for DN. PMID:25003128

  9. L-Ornithine Schiff base-copper and -cadmium complexes as new proteasome inhibitors and apoptosis inducers in human cancer cells.

    PubMed

    Zhang, Zhongyu; Bi, Caifeng; Fan, Yuhua; Zhang, Nan; Deshmukh, Rahul; Yan, Xingchen; Lv, Xiuwen; Zhang, Pengfei; Zhang, Xia; Dou, Q Ping

    2015-01-01

    Ubiquitin-proteasome system (UPS) plays a crucial role in many cellular processes such as cell cycle, proliferation and apoptosis. Aberrant activation of UPS may result in cellular transformation or other altered pathological conditions. Previous studies have shown that metal-based complexes could inhibit proteasome activity and induce apoptosis in certain human cancer cells. In the current study, we report that the cadmium and copper complexes with heterocycle-ornithine Schiff base are potent inhibitors of proteasomal chymotrypsin-like (CT-like) activity, leading to induction of apoptosis in cancer cells. Two novel copper-containing complexes and two novel cadmium-containing complexes with different heterocycle-ornithine Schiff base structures as ligands were synthesized and characterized. We found that complexes Cu1, Cd1 and Cd2 show proteasome-inhibitory activities in human breast cancer MDA-MB-231 and human prostate cancer LNCaP cells, resulting in the accumulation of p27, a natural proteasome substrate and other ubiquitinated proteins, followed by the induction of apoptosis. Our results suggest that metal complexes with heterocycle-ornithine Schiff base have proteasome-inhibitory capabilities and have the potential to be developed into novel anticancer drugs. PMID:25467055

  10. Proteasome Inhibitor Bortezomib Suppresses Nuclear Factor-Kappa B Activation and Ameliorates Eye Inflammation in Experimental Autoimmune Uveitis

    PubMed Central

    Hsu, Sheng-Min; Yang, Chang-Hao; Shen, Fang-Hsiu; Chen, Shun-Hua; Lin, Chia-Jhen; Shieh, Chi-Chang

    2015-01-01

    Bortezomib is a proteasome inhibitor used for hematologic cancer treatment. Since it can suppress NF-κB activation, which is critical for the inflammatory process, bortezomib has been found to possess anti-inflammatory activity. In this study, we evaluated the effect of bortezomib on experimental autoimmune uveitis (EAU) in mice and investigated the potential mechanisms related to NF-κB inactivation. High-dose bortezomib (0.75 mg/kg), low-dose bortezomib (0.15 mg/kg), or phosphate buffered saline was given after EAU induction. We found that the EAU is ameliorated by high-dose bortezomib treatment when compared with low-dose bortezomib or PBS treatment. The DNA-binding activity of NF-κB was suppressed and expression of several key inflammatory mediators including TNF-α, IL-1α, IL-1β, IL-12, IL-17, and MCP-1 was lowered in the high-dose bortezomib-treated group. These results suggest that proteasome inhibition is a promising treatment strategy for autoimmune uveitis. PMID:25653480

  11. Phase 1 study of weekly dosing with the investigational oral proteasome inhibitor ixazomib in relapsed/refractory multiple myeloma.

    PubMed

    Kumar, Shaji K; Bensinger, William I; Zimmerman, Todd M; Reeder, Craig B; Berenson, James R; Berg, Deborah; Hui, Ai-Min; Gupta, Neeraj; Di Bacco, Alessandra; Yu, Jiang; Shou, Yaping; Niesvizky, Ruben

    2014-08-14

    Proteasome inhibition is an effective treatment strategy for multiple myeloma. With improving survival, attention is increasingly focusing on ease of administration and toxicity profile. Ixazomib is an investigational, orally bioavailable 20S proteasome inhibitor. Sixty patients with relapsed and/or refractory multiple myeloma were enrolled on this phase 1 trial to evaluate safety and tolerability and determine the maximum tolerated dose (MTD) of single-agent, oral ixazomib given weekly for 3 of 4 weeks. Upon MTD determination, patients were enrolled to 4 different cohorts based on relapsed/refractory status and prior bortezomib and carfilzomib exposure. The MTD was determined to be 2.97 mg/m(2). Dose-limiting toxicities were grade 3 nausea, vomiting, and diarrhea in 2 patients, and grade 3 skin rash in 1 patient. Common drug-related adverse events were thrombocytopenia (43%), diarrhea (38%), nausea (38%), fatigue (37%), and vomiting (35%). The observed rate of peripheral neuropathy was 20%, with only 1 grade 3 event reported. Nine (18%) patients achieved a partial response or better, including 8 of 30 (27%) evaluable patients treated at the MTD. Pharmacokinetic studies suggested a long terminal half-life of 3.6 to 11.3 days, supporting once-weekly dosing. This trial was registered at www.clinicaltrials.gov as #NCT00963820. PMID:24904120

  12. Phase 1 study of weekly dosing with the investigational oral proteasome inhibitor ixazomib in relapsed/refractory multiple myeloma

    PubMed Central

    Bensinger, William I.; Zimmerman, Todd M.; Reeder, Craig B.; Berenson, James R.; Berg, Deborah; Hui, Ai-Min; Gupta, Neeraj; Di Bacco, Alessandra; Yu, Jiang; Shou, Yaping; Niesvizky, Ruben

    2014-01-01

    Proteasome inhibition is an effective treatment strategy for multiple myeloma. With improving survival, attention is increasingly focusing on ease of administration and toxicity profile. Ixazomib is an investigational, orally bioavailable 20S proteasome inhibitor. Sixty patients with relapsed and/or refractory multiple myeloma were enrolled on this phase 1 trial to evaluate safety and tolerability and determine the maximum tolerated dose (MTD) of single-agent, oral ixazomib given weekly for 3 of 4 weeks. Upon MTD determination, patients were enrolled to 4 different cohorts based on relapsed/refractory status and prior bortezomib and carfilzomib exposure. The MTD was determined to be 2.97 mg/m2. Dose-limiting toxicities were grade 3 nausea, vomiting, and diarrhea in 2 patients, and grade 3 skin rash in 1 patient. Common drug-related adverse events were thrombocytopenia (43%), diarrhea (38%), nausea (38%), fatigue (37%), and vomiting (35%). The observed rate of peripheral neuropathy was 20%, with only 1 grade 3 event reported. Nine (18%) patients achieved a partial response or better, including 8 of 30 (27%) evaluable patients treated at the MTD. Pharmacokinetic studies suggested a long terminal half-life of 3.6 to 11.3 days, supporting once-weekly dosing. This trial was registered at www.clinicaltrials.gov as #NCT00963820. PMID:24904120

  13. Intracellular colocalization of HAP1/STBs with steroid hormone receptors and its enhancement by a proteasome inhibitor

    SciTech Connect

    Fujinaga, Ryutaro; Takeshita, Yukio; Yoshioka, Kazuhiro; Nakamura, Hiroyuki; Shinoda, Shuhei; Islam, Md. Nabiul; Jahan, Mir Rubayet; Yanai, Akie; Kokubu, Keiji; Shinoda, Koh

    2011-07-15

    The stigmoid body (STB) is a cytoplasmic inclusion containing huntingtin-associated protein 1 (HAP1), and HAP1/STB formation is induced by transfection of the HAP1 gene into cultured cells. In the present study, we examined the intracellular colocalization of HAP1/STBs with steroid hormone receptors (SHRs), including the androgen receptor (AR), estrogen receptor, glucocorticoid receptor (GR), and mineralocorticoid receptor, in COS-7 cells cotransfected with HAP1 and each receptor. We found that C-terminal ligand-binding domains of all SHRs had potential for colocalization with HAP1/STBs, whereas only AR and GR were clearly colocalized with HAP1/STBs when each full-length SHR was coexpressed with HAP1. In addition, it appeared that HAP1/STBs did not disrupt GR and AR functions because the receptors on HAP1/STBs maintained nuclear translocation activity in response to their specific ligands. When the cells were treated with a proteasome inhibitor, GR and AR localized outside HAP1/STBs translocated into the nucleus, whereas the receptors colocalized with HAP1/STBs persisted in their colocalization even after treatment with their ligands. Therefore, HAP1/STBs may be involved in cytoplasmic modifications of the nuclear translocation of GR and AR in a ubiquitin-proteasome system.

  14. Generating a Generation of Proteasome Inhibitors: From Microbial Fermentation to Total Synthesis of Salinosporamide A (Marizomib) and Other Salinosporamides

    PubMed Central

    Potts, Barbara C.; Lam, Kin S.

    2010-01-01

    The salinosporamides are potent proteasome inhibitors among which the parent marine-derived natural product salinosporamide A (marizomib; NPI-0052; 1) is currently in clinical trials for the treatment of various cancers. Methods to generate this class of compounds include fermentation and natural products chemistry, precursor-directed biosynthesis, mutasynthesis, semi-synthesis, and total synthesis. The end products range from biochemical tools for probing mechanism of action to clinical trials materials; in turn, the considerable efforts to produce the target molecules have expanded the technologies used to generate them. Here, the full complement of methods is reviewed, reflecting remarkable contributions from scientists of various disciplines over a period of 7 years since the first publication of the structure of 1. PMID:20479958

  15. Perturbation of Hsp90 interaction with nascent CFTR prevents its maturation and accelerates its degradation by the proteasome.

    PubMed Central

    Loo, M A; Jensen, T J; Cui, L; Hou, Y; Chang, X B; Riordan, J R

    1998-01-01

    Maturation of wild-type CFTR nascent chains at the endoplasmic reticulum (ER) occurs inefficiently; many disease-associated mutant forms do not mature but instead are eliminated by proteolysis involving the cytosolic proteasome. Although calnexin binds nascent CFTR via its oligosaccharide chains in the ER lumen and Hsp70 binds CFTR cytoplasmic domains, perturbation of these interactions alone is without major influence on maturation or degradation. We show that the ansamysin drugs, geldanamycin and herbimycin A, which inhibit the assembly of some signaling molecules by binding to specific sites on Hsp90 in the cytosol or Grp94 in the ER lumen, block the maturation of nascent CFTR and accelerate its degradation. The immature CFTR molecule was detected in association with Hsp90 but not with Grp94, and geldanamycin prevented the Hsp90 association. The drug-enhanced degradation was decreased by lactacystin and other proteasome inhibitors. Therefore, consistent with other examples of countervailing effects of Hsp90 and the proteasome, it would seem that this chaperone may normally contribute to CFTR folding and, when this function is interfered with by an ansamycin, there is a further shift to proteolytic degradation. This is the first direct evidence of a role for Hsp90 in the maturation of a newly synthesized integral membrane protein by interaction with its cytoplasmic domains on the ER surface. PMID:9843494

  16. Perturbation of Hsp90 interaction with nascent CFTR prevents its maturation and accelerates its degradation by the proteasome.

    PubMed

    Loo, M A; Jensen, T J; Cui, L; Hou, Y; Chang, X B; Riordan, J R

    1998-12-01

    Maturation of wild-type CFTR nascent chains at the endoplasmic reticulum (ER) occurs inefficiently; many disease-associated mutant forms do not mature but instead are eliminated by proteolysis involving the cytosolic proteasome. Although calnexin binds nascent CFTR via its oligosaccharide chains in the ER lumen and Hsp70 binds CFTR cytoplasmic domains, perturbation of these interactions alone is without major influence on maturation or degradation. We show that the ansamysin drugs, geldanamycin and herbimycin A, which inhibit the assembly of some signaling molecules by binding to specific sites on Hsp90 in the cytosol or Grp94 in the ER lumen, block the maturation of nascent CFTR and accelerate its degradation. The immature CFTR molecule was detected in association with Hsp90 but not with Grp94, and geldanamycin prevented the Hsp90 association. The drug-enhanced degradation was decreased by lactacystin and other proteasome inhibitors. Therefore, consistent with other examples of countervailing effects of Hsp90 and the proteasome, it would seem that this chaperone may normally contribute to CFTR folding and, when this function is interfered with by an ansamycin, there is a further shift to proteolytic degradation. This is the first direct evidence of a role for Hsp90 in the maturation of a newly synthesized integral membrane protein by interaction with its cytoplasmic domains on the ER surface. PMID:9843494

  17. Hypoxia and hypoxia mimetics decrease aquaporin 5 (AQP5) expression through both hypoxia inducible factor-1α and proteasome-mediated pathways.

    PubMed

    Kawedia, Jitesh D; Yang, Fan; Sartor, Maureen A; Gozal, David; Czyzyk-Krzeska, Maria; Menon, Anil G

    2013-01-01

    The alveolar epithelium plays a central role in gas exchange and fluid transport, and is therefore critical for normal lung function. Since the bulk of water flux across this epithelium depends on the membrane water channel Aquaporin 5 (AQP5), we asked whether hypoxia had any effect on AQP5 expression. We show that hypoxia causes a significant (70%) decrease in AQP5 expression in the lungs of mice exposed to hypoxia. Hypoxia and the hypoxia mimetic, cobalt, also caused similar decreases in AQP5 mRNA and protein expression in the mouse lung epithelial cell line MLE-12. The action of hypoxia and cobalt on AQP5 transcription was demonstrated by directly quantifying heternonuclear RNA by real-time PCR. Dominant negative mutants of Hypoxia Inducible Factor (HIF-1α) and HIF-1α siRNA blocked the action of cobalt, showing that HIF-1α is a key component in this mechanism. The proteasome inhibitors, lactacystin or proteasome inhibitor-III completely abolished the effect of hypoxia and cobalt both at the protein and mRNA level indicating that the proteasome pathway is probably involved not only for the stability of HIF-1α protein, but for the stability of unidentified transcription factors that regulate AQP5 transcription. These studies reveal a potentially important physiological mechanism linking hypoxic stress and membrane water channels. PMID:23469202

  18. Combination Treatment with Sublethal Ionizing Radiation and the Proteasome Inhibitor, Bortezomib, Enhances Death-Receptor Mediated Apoptosis and Anti-Tumor Immune Attack

    PubMed Central

    Cacan, Ercan; Spring, Alexander M.; Kumari, Anita; Greer, Susanna F.; Garnett-Benson, Charlie

    2015-01-01

    Sub-lethal doses of radiation can modulate gene expression, making tumor cells more susceptible to T-cell-mediated immune attack. Proteasome inhibitors demonstrate broad anti-tumor activity in clinical and pre-clinical cancer models. Here, we use a combination treatment of proteasome inhibition and irradiation to further induce immunomodulation of tumor cells that could enhance tumor-specific immune responses. We investigate the effects of the 26S proteasome inhibitor, bortezomib, alone or in combination with radiotherapy, on the expression of immunogenic genes in normal colon and colorectal cancer cell lines. We examined cells for changes in the expression of several death receptors (DR4, DR5 and Fas) commonly used by T cells for killing of target cells. Our results indicate that the combination treatment resulted in increased cell surface expression of death receptors by increasing their transcript levels. The combination treatment further increases the sensitivity of carcinoma cells to apoptosis through FAS and TRAIL receptors but does not change the sensitivity of normal non-malignant epithelial cells. Furthermore, the combination treatment significantly enhances tumor cell killing by tumor specific CD8+ T cells. This study suggests that combining radiotherapy and proteasome inhibition may simultaneously enhance tumor immunogenicity and the induction of antitumor immunity by enhancing tumor-specific T-cell activity. PMID:26703577

  19. Effects of proteasome inhibitors MG132, ZL3VS and AdaAhx3L3VS on protein metabolism in septic rats

    PubMed Central

    Kadlčíková, Jana; Holeček, Milan; Šafránek, Roman; Tilšer, Ivan; Kessler, Benedikt M

    2004-01-01

    Proteasome inhibitors are novel therapeutic agents for the treatment of cancer and other severe disorders. One of the possible side effects is influencing the metabolism of proteins. The aim of our study was to evaluate the influence of three proteasome inhibitors MG132, ZL3VS and AdaAhx3L3VS on protein metabolism and leucine oxidation in incubated skeletal muscle of control and septic rats. Total proteolysis was determined according to the rates of tyrosine release into the medium during incubation. The rates of protein synthesis and leucine oxidation were measured in a medium containing L-[1-14C]leucine. Protein synthesis was determined as the amount of L-[1-14C]leucine incorporated into proteins, and leucine oxidation was evaluated according to the release of 14CO2 during incubation. Sepsis was induced in rats by means of caecal ligation and puncture. MG132 reduced proteolysis by more than 50% and protein synthesis by 10–20% in the muscles of healthy rats. In septic rats, proteasome inhibitors, except ZL3VS, decreased proteolysis in both soleus and extensor digitorum longus (EDL) muscles, although none of the inhibitors had any effect on protein synthesis. Leucine oxidation was increased by AdaAhx3L3VS in the septic EDL muscle and decreased by MG132 in intact EDL muscle. We conclude that MG132 and AdaAhx3L3VS reversed protein catabolism in septic rat muscles. PMID:15566433

  20. Identification of noncovalent proteasome inhibitors with high selectivity for chymotrypsin-like activity by a multistep structure-based virtual screening.

    PubMed

    Di Giovanni, Carmen; Ettari, Roberta; Sarno, Serena; Rotondo, Archimede; Bitto, Alessandra; Squadrito, Francesco; Altavilla, Domenica; Schirmeister, Tanja; Novellino, Ettore; Grasso, Silvana; Zappalà, Maria; Lavecchia, Antonio

    2016-10-01

    Noncovalent proteasome inhibitors introduce an alternative mechanism of inhibition to that of covalent inhibitors, e.g. carfilzomib, used in cancer therapy. A multistep hierarchical structure-based virtual screening (SBVS) of the 65,375 NCI lead-like compound library led to the identification of two compounds (9 and 28) which noncovalently inhibited the chymotrypsin-like (ChT-L) activity (Ki = 2.18 and 2.12 μM, respectively) with little or no effects on the other two major proteasome proteolytic activities, trypsin-like (T-L) and post-glutamyl peptide hydrolase (PGPH) activities. A subsequent hierarchical similarity search over the full NCI database with the most active tripeptide-based inhibitor 9 resulted in the discovery of the β5/β6-specific tripeptide derivative 38 that noncovalently binds the ChT-L site (Ki = 0.42 μM). The solution structure of 9 and 38 was solved by (1)H NMR spectroscopy and the binding mode of the inhibitors was elucidated by docking experiments using the yeast 20S proteasome. Compound 38 (IC50 = 26.7 μM) is slightly more potent than 9 (IC50 = 34.3 μM) at inhibiting survival of dexamethasone-resistant (MM.1R) human multiple myeloma cells. The identified ligand thus provides valuable insights for the future structure-based design of subtype-specific proteasome inhibitors. PMID:27318981

  1. A UHPLC-UV-QTOF study on the stability of carfilzomib, a novel proteasome inhibitor.

    PubMed

    Sestak, Vit; Roh, Jaroslav; Klepalova, Libuse; Kovarikova, Petra

    2016-05-30

    This study addresses the lack of data on the stability of carfilzomib, a newly approved proteasome-inhibiting anticancer drug. A new stability-indicating UHPLC-UV method for analysis of carfilzomib was developed and validated within the concentrations of 10-250 μg/mL. The aforementioned method was utilized to evaluate the effects of forced degradation and to investigate the degradation kinetics, as well as to examine drug stability in a pharmaceutical formulation. A UHPLC-QTOF method was utilized to identify the principal degradation products. It was found that carfilzomib: (1) is stable at neutral and slightly acidic pH, but prone to degradation at both high and low pH; (2) is acceptably stable in the pharmaceutical formulation; but (3) is prone to oxidation and photodegradation. Carfilzomib degradation followed first-order kinetics. The decomposition products resulted from peptide bond hydrolysis, epoxide hydrolysis, hydrogen chloride addition, base-catalyzed Robinson-Gabriel reaction, tertiary amine oxidation and isomerization. Our results document, for the first time, the inherent stability of carfilzomib and provide information about the identity of its degradation products. These results highlight the stability issues that need to be kept in mind for handling and storage of carfilzomib. PMID:26994320

  2. Accumulation of heme oxygenase-1 (HSP32) in Xenopus laevis A6 kidney epithelial cells treated with sodium arsenite, cadmium chloride or proteasomal inhibitors.

    PubMed

    Music, Ena; Khan, Saad; Khamis, Imran; Heikkila, John J

    2014-11-01

    The present study examined the effect of sodium arsenite, cadmium chloride, heat shock and the proteasomal inhibitors MG132, withaferin A and celastrol on heme oxygenase-1 (HO-1; also known as HSP32) accumulation in Xenopus laevis A6 kidney epithelial cells. Immunoblot analysis revealed that HO-1 accumulation was not induced by heat shock but was enhanced by sodium arsenite and cadmium chloride in a dose- and time-dependent fashion. Immunocytochemistry revealed that these metals induced HO-1 accumulation in a granular pattern primarily in the cytoplasm. Additionally, in 20% of the cells arsenite induced the formation of large HO-1-containing perinuclear structures. In cells recovering from sodium arsenite or cadmium chloride treatment, HO-1 accumulation initially increased to a maximum at 12h followed by a 50% reduction at 48 h. This initial increase in HO-1 levels was likely the result of new synthesis as it was inhibited by cycloheximide. Interestingly, treatment of cells with a mild heat shock enhanced HO-1 accumulation induced by low concentrations of sodium arsenite and cadmium chloride. Finally, we determined that HO-1 accumulation was induced in A6 cells by the proteasomal inhibitors, MG132, withaferin A and celastrol. An examination of heavy metal and proteasomal inhibitor-induced HO-1 accumulation in amphibians is of importance given the presence of toxic heavy metals in aquatic habitats. PMID:25064141

  3. Phase 1 dose-escalation study of IV ixazomib, an investigational proteasome inhibitor, in patients with relapsed/refractory lymphoma.

    PubMed

    Assouline, S E; Chang, J; Cheson, B D; Rifkin, R; Hamburg, S; Reyes, R; Hui, A-M; Yu, J; Gupta, N; Di Bacco, A; Shou, Y; Martin, P

    2014-01-01

    Ixazomib is an investigational proteasome inhibitor that has shown preclinical activity in lymphoma models. This phase 1 study assessed the safety, tolerability, maximum tolerated dose (MTD), pharmacokinetics, pharmacodynamics and preliminary activity of intravenous (IV) ixazomib in relapsed/refractory lymphoma patients who had received ⩾ 2 prior therapies. Thirty patients with a range of histologies received ixazomib 0.125-3.11 mg/m(2) on days 1, 8 and 15 of 28-day cycles. Patients received a median of two cycles (range 1-36). MTD was determined to be 2.34 mg/m(2). Most common drug-related adverse events (AEs) included fatigue (43%), diarrhea (33%), nausea, vomiting and thrombocytopenia (each 27%). Drug-related grade ⩾ 3 AEs included neutropenia (20%), thrombocytopenia (13%) and diarrhea (10%). Drug-related peripheral neuropathy occurred in four (13%) patients; no grade ⩾ 3 events were reported. Plasma exposure increased dose proportionally from 0.5-3.11 mg/m(2); terminal half-life was 4-12 days after multiple dosing. Of 26 evaluable patients, five achieved responses: 4/11 follicular lymphoma patients (one complete and three partial responses) and 1/4 peripheral T-cell lymphoma patients (partial response). Sustained responses were observed with ⩾ 32 cycles of treatment in two heavily pretreated follicular lymphoma patients. Results suggest weekly IV ixazomib is generally well tolerated and may be clinically active in relapsed/refractory lymphoma. PMID:25325301

  4. Phase 1 dose-escalation study of IV ixazomib, an investigational proteasome inhibitor, in patients with relapsed/refractory lymphoma

    PubMed Central

    Assouline, S E; Chang, J; Cheson, B D; Rifkin, R; Hamburg, S; Reyes, R; Hui, A-M; Yu, J; Gupta, N; Di Bacco, A; Shou, Y; Martin, P

    2014-01-01

    Ixazomib is an investigational proteasome inhibitor that has shown preclinical activity in lymphoma models. This phase 1 study assessed the safety, tolerability, maximum tolerated dose (MTD), pharmacokinetics, pharmacodynamics and preliminary activity of intravenous (IV) ixazomib in relapsed/refractory lymphoma patients who had received ⩾2 prior therapies. Thirty patients with a range of histologies received ixazomib 0.125−3.11 mg/m2 on days 1, 8 and 15 of 28-day cycles. Patients received a median of two cycles (range 1−36). MTD was determined to be 2.34 mg/m2. Most common drug-related adverse events (AEs) included fatigue (43%), diarrhea (33%), nausea, vomiting and thrombocytopenia (each 27%). Drug-related grade ⩾3 AEs included neutropenia (20%), thrombocytopenia (13%) and diarrhea (10%). Drug-related peripheral neuropathy occurred in four (13%) patients; no grade ⩾3 events were reported. Plasma exposure increased dose proportionally from 0.5−3.11 mg/m2; terminal half-life was 4−12 days after multiple dosing. Of 26 evaluable patients, five achieved responses: 4/11 follicular lymphoma patients (one complete and three partial responses) and 1/4 peripheral T-cell lymphoma patients (partial response). Sustained responses were observed with ⩾32 cycles of treatment in two heavily pretreated follicular lymphoma patients. Results suggest weekly IV ixazomib is generally well tolerated and may be clinically active in relapsed/refractory lymphoma. PMID:25325301

  5. Obatoclax interacts synergistically with the irreversible proteasome inhibitor carfilzomib in GC- and ABC- DLBCL cells in vitro and in vivo

    PubMed Central

    Dasmahapatra, Girija; Lembersky, Dmitry; Son, Minkyeong P.; Patel, Hiral; Peterson, Derick; Attkisson, Elisa; Fisher, Richard I.; Friedberg, Jonathan W.; Dent, Paul; Grant, Steven

    2013-01-01

    Interactions between the the irreversible proteasome inhibitor carfilzomib (CFZ) and the pan-BH3 mimetic obatoclax (Obato) were examined in GC- and ABC-DLBCL cells. Co-treatment with minimally toxic concentrations of CFZ (i.e., 2–6 nM) and sub-toxic concentrations of obato (0.05–2.0μM) synergistically increased apoptosis in multiple DLBCL cell lines and increased lethality toward primary human DLBCL but not normal CD34+ cells. Synergistic interactions were associated with sharp increases in caspase-3 activation, PARP cleavage, phospho-JNK induction, up-regulation of Noxa, and AKT dephosphorylation. Combined treatment also diminished CFZ-mediated Mcl-1 up-regulation while immunoprecipitation analysis revealed reduced associations between Bak and Mcl-1/Bcl-xL, and Bim and Mcl-1. The CFZ/Obato regimen triggered translocation, conformational change and dimerization of Bax and activation of Bak. Genetic interruption of JNK and Noxa by shRNA knockdown, ectopic Mcl-1 expression, or enforced activation of AKT significantly attenuated CFZ/Obato-mediated apoptosis. Notably, co-administration of CFZ/Obato sharply increased apoptosis in multiple bortezomib-resistant DLBCL models. Finally, in vivo administration of CFZ and Obato to mice inoculated with SUDHL4 cells substantially suppressed tumor growth, activated JNK, inactivated AKT, and increased survival compared to the effects of single agent treatment. Together, these findings argue that a strategy combining CFZ and Obato warrants attention in DLBCL. PMID:22411899

  6. Treatment with the HIV protease inhibitor nelfinavir triggers the unfolded protein response and may overcome proteasome inhibitor resistance of multiple myeloma in combination with bortezomib: a phase I trial (SAKK 65/08)

    PubMed Central

    Driessen, Christoph; Kraus, Marianne; Joerger, Markus; Rosing, Hilde; Bader, Jürgen; Hitz, Felicitas; Berset, Catherine; Xyrafas, Alexandros; Hawle, Hanne; Berthod, Gregoire; Overkleeft, Hermann S.; Sessa, Christiana; Huitema, Alwin; Pabst, Thomas; von Moos, Roger; Hess, Dagmar; Mey, Ulrich J.M.

    2016-01-01

    Downregulation of the unfolded protein response mediates proteasome inhibitor resistance in multiple myeloma. The Human Immunodeficieny Virus protease inhibitor nelfinavir activates the unfolded protein response in vitro. We determined dose-limiting toxicity and recommended dose for phase II of nelfinavir in combination with the proteasome inhibitor bortezomib. Twelve patients with advanced hematologic malignancies were treated with nelfinavir (2500–5000 mg/day p.o., days 1–14, 3+3 dose escalation) and bortezomib (1.3 mg/m2, days 1, 4, 8, 11; 21-day cycles). A run in phase with nelfinavir monotherapy allowed pharmakokinetic/pharmakodynamic assessment of nelfinavir in the presence or absence of concomittant bortezomib. End points included dose-limiting toxicity, activation of the unfolded protein response, proteasome activity, toxicity and response to trial treatment. Nelfinavir 2×2500 mg was the recommended phase II dose identified. Nelfinavir alone significantly up-regulated expression of proteins related to the unfolded protein response in peripheral blood mononuclear cells and inhibited proteasome activity. Of 10 evaluable patients in the dose escalation cohort, 3 achieved a partial response, 4 stable disease for 2 cycles or more, while 3 had progressive disease as best response. In an exploratory extension cohort with 6 relapsed, bortezomib-refractory, lenalidomide-resistant myeloma patients treated at the recommended phase II dose, 3 reached a partial response, 2 a minor response, and one progressive disease. The combination of nelfinavir with bortezomib is safe and shows promising activity in advanced, bortezomib-refractory multiple myeloma. Induction of the unfolded protein response by nelfinavir may overcome the biological features of proteasome inhibitor resistance. PMID:26659919

  7. Incorporation of Non-natural Amino Acids Improves Cell Permeability and Potency of Specific Inhibitors of Proteasome Trypsin-like Sites

    PubMed Central

    Geurink, Paul P.; van der Linden, Wouter A.; Mirabella, Anne C.; Gallastegui, Nerea; de Bruin, Gerjan; Blom, Annet E. M.; Voges, Mathias J.; Mock, Elliot D.; Florea, Bogdan I.; van der Marel, Gijs A.; Driessen, Christoph; van der Stelt, Mario; Groll, Michael; Overkleeft, Herman S.; Kisselev, Alexei F.

    2013-01-01

    Proteasomes degrade the majority of proteins in mammalian cells by a concerted action of three distinct pairs of active sites. The chymotrypsin-like sites are targets of antimyeloma agents bortezomib and carfilzomib. Inhibitors of the trypsin-like site sensitize multiple myeloma cells to these agents. Here we describe systematic effort to develop inhibitors with improved potency and cell permeability, yielding azido-Phe-Leu-Leu-4-aminomethyl-Phe-methyl vinyl sulfone (4a, LU-102), and a fluorescent activity-based probe for this site. X-ray structures of 4a and related inhibitors complexed with yeast proteasomes revealed the structural basis for specificity. Nontoxic to myeloma cells when used as a single agent, 4a sensitized them to bortezomib and carfilzomib. This sensitizing effect was much stronger than the synergistic effects of histone acetylase inhibitors or additive effects of doxorubicin and dexamethasone, raising the possibility that combinations of inhibitors of the trypsin-like site with bortezomib or carfilzomib would have stronger antineoplastic activity than combinations currently used clinically. PMID:23320547

  8. Bacterial Proteasomes

    PubMed Central

    Jastrab, Jordan B.; Darwin, K. Heran

    2015-01-01

    Interest in bacterial proteasomes was sparked by the discovery that proteasomal degradation is required for the pathogenesis of Mycobacterium tuberculosis, one of the world's deadliest pathogens. Although bacterial proteasomes are structurally similar to their eukaryotic and archaeal homologs, there are key differences in their mechanisms of assembly, activation, and substrate targeting for degradation. In this article, we compare and contrast bacterial proteasomes with their archaeal and eukaryotic counterparts, and we discuss recent advances in our understanding of how bacterial proteasomes function to influence microbial physiology. PMID:26488274

  9. Resistance Gene-Guided Genome Mining: Serial Promoter Exchanges in Aspergillus nidulans Reveal the Biosynthetic Pathway for Fellutamide B, a Proteasome Inhibitor.

    PubMed

    Yeh, Hsu-Hua; Ahuja, Manmeet; Chiang, Yi-Ming; Oakley, C Elizabeth; Moore, Shauna; Yoon, Olivia; Hajovsky, Heather; Bok, Jin-Woo; Keller, Nancy P; Wang, Clay C C; Oakley, Berl R

    2016-08-19

    Fungal genome projects are revealing thousands of cryptic secondary metabolism (SM) biosynthetic gene clusters that encode pathways that potentially produce valuable compounds. Heterologous expression systems should allow these clusters to be expressed and their products obtained, but approaches are needed to identify the most valuable target clusters. The inp cluster of Aspergillus nidulans contains a gene, inpE, that encodes a proteasome subunit, leading us to hypothesize that the inp cluster produces a proteasome inhibitor and inpE confers resistance to this compound. Previous efforts to express this cluster have failed, but by sequentially replacing the promoters of the genes of the cluster with a regulatable promotor, we have expressed them successfully. Expression reveals that the product of the inp cluster is the proteasome inhibitor fellutamide B, and our data allow us to propose a biosynthetic pathway for the compound. By deleting inpE and activating expression of the inp cluster, we demonstrate that inpE is required for resistance to internally produced fellutamide B. These data provide experimental validation for the hypothesis that some fungal SM clusters contain genes that encode resistant forms of the enzymes targeted by the compound produced by the cluster. PMID:27294372

  10. LDL suppresses angiogenesis through disruption of the HIF pathway via NF-κB inhibition which is reversed by the proteasome inhibitor BSc2118

    PubMed Central

    Doeppner, Thorsten R.; Niu, Feng; Li, Qiaochuan; Yang, Yanping; Kuckelkorn, Ulrike; Hagemann, Nina; Li, Wei; Hermann, Dirk M.; Dai, Yun; Zhou, Wen; Jin, Fengyan

    2015-01-01

    Since disturbance of angiogenesis predisposes to ischemic injuries, attempts to promote angiogenesis have been made to improve clinical outcomes of patients with many ischemic disorders. While hypoxia inducible factors (HIFs) stimulate vascular remodeling and angiogenesis, hyperlipidemia impairs angiogenesis in response to various pro-angiogenic factors. However, it remains uncertain how HIFs regulate angiogenesis under hyperlipidemia. Here, we report that exposure to low-density lipoprotein (LDL) suppressed in vitro angiogenesis of human brain microvascular endothelial cells. Whereas LDL exposure diminished expression of HIF-1α and HIF-2α induced by hypoxia, it inhibited DMOG- and TNFα-induced HIF-1α and HIF-2α expression in normoxia. Notably, in both hypoxia and normoxia, LDL markedly reduced expression of HIF-1β, a constitutively stable HIF subunit, an event associated with NF-κB inactivation. Moreover, knockdown of HIF-1β down-regulated HIF-1α and HIF-2α expression, in association with increased HIF-1α hydroxylation and 20S proteasome activity after LDL exposure. Significantly, the proteasome inhibitor BSc2118 prevented angiogenesis attenuation by LDL through restoring expression of HIFs. Together, these findings argue that HIF-1β might act as a novel cross-link between the HIF and NF-κB pathways in suppression of angiogenesis by LDL, while proteasome inhibitors might promote angiogenesis by reactivating this signaling cascade under hyperlipidemia. PMID:26388611

  11. A binuclear complex constituted by diethyldithiocarbamate and copper(I) functions as a proteasome activity inhibitor in pancreatic cancer cultures and xenografts

    SciTech Connect

    Han, Jinbin; Yue, Xiaoqiang; Chang, Jinjia; Shi, Weidong; Hua, Yongqiang

    2013-12-15

    It is a therapeutic strategy for cancers including pancreatic to inhibit proteasome activity. Disulfiram (DSF) may bind copper (Cu) to form a DSF–Cu complex. DSF–Cu is capable of inducing apoptosis in cancer cells by inhibiting proteasome activity. DSF is rapidly converted to diethyldithiocarbamate (DDTC) within bodies. Copper(II) absorbed by bodies is reduced to copper(I) when it enters cells. We found that DDTC and copper(I) could form a binuclear complex which might be entitled DDTC–Cu(I), and it had been synthesized by us in the laboratory. This study is to investigate the anticancer potential of this complex on pancreatic cancer and the possible mechanism. Pancreatic cancer cell lines, SW1990, PANC-1 and BXPC-3 were used for in vitro assays. Female athymic nude mice grown SW1990 xenografts were used as animal models. Cell counting kit-8 (cck-8) assay and flow cytometry were used for analyzing apoptosis in cells. A 20S proteasome assay kit was used in proteasome activity analysis. Western blot (WB) and immunohistochemistry (IHC) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were used in tumor sample analysis. The results suggest that DDTC–Cu(I) inhibit pancreatic cancer cell proliferation and proteasome activity in vitro and in vivo. Accumulation of ubiquitinated proteins, and increased p27 as well as decreased NF-κB expression were detected in tumor tissues of DDTC–Cu(I)-treated group. Our data indicates that DDTC–Cu(I) is an effective proteasome activity inhibitor with the potential to be explored as a drug for pancreatic cancer. - Highlights: • A new structure of DDTC–Cu(I) was reported for the first time. • DDTC–Cu(I) dissolved directly in water was for in vitro and in vivo uses. • DDTC–Cu(I) demonstrated significant anticancer effect in vitro and in vivo. • DDTC–Cu(I) is capable of inhibiting proteasome activity in vitro and in vivo.

  12. Comparison of antiproliferative and apoptotic effects of a novel proteasome inhibitor MLN2238 with bortezomib on K562 chronic myeloid leukemia cells.

    PubMed

    Engür, Selin; Dikmen, Miriş; Öztürk, Yusuf

    2016-01-01

    Inhibition of the proteasome has emerged as a clinically effective anticancer therapeutic approach in recent years. Bortezomib (Velcade®) showed extremely high potency against a wide range of cancer cell lines. Ixazomib (MLN9708-MLN2238), the second-generation proteasome inhibitor, selectivity and potency were similar to that of bortezomib, is currently being investigated in phase I studies. It shows superior antitumor activity in hematologic malignancy, especially multiple myelomas. In this study, for the first time, we evaluated and compared the antiproliferative and apoptotic effects of the novel proteasome inhibitor MLN2238 (the active form of MLN9708) with bortezomib using in vitro chronic myeloid leukemia. Cytotoxic and apoptotic effects of MLN2238 and bortezomib were determined by trypan blue dye exclusion assays, WST-1 cell proliferation assay, increased AnnexinV-PI binding capacity, changes in caspase-3 activity and loss of mitochondrial membrane potential (JC-1). Associated with proteasome pathway NFκB1 and c-myc mRNA expression levels were examined by the qRT-PCR method. We observed that cytotoxic and apoptotic effects on K562 cells were started at 5 μm of MLN2238 and 1 μm of bortezomib after 24 and 48 h. Also, MLN2238 and bortezomib downregulated NFκB1 and c-myc mRNA expression at 24 h. Our result revealed that MLN22238 and bortezomib had significant cytotoxic and apoptotic effects on K562 cells. Here, we first demonstrate in vitro data that support the development of MLN2238, by direct comparison with bortezomib on K562 cells. PMID:26667773

  13. Negative feedback regulation of NF-κB-inducing kinase is proteasome-dependent but does not require cellular inhibitors of apoptosis.

    PubMed

    Gray, Carolyn M; McCorkell, Kelly A; Chunduru, Srinivas K; McKinlay, Mark A; May, Michael J

    2014-07-18

    Non-canonical NF-κB signaling is controlled by the precise regulation of NF-κB inducing kinase (NIK) stability. NIK is constitutively ubiquitylated by cellular inhibitor of apoptosis (cIAP) proteins 1 and 2, leading to its complete proteasomal degradation in resting cells. Following stimulation, cIAP-mediated ubiquitylation of NIK ceases and NIK is stabilized, allowing for inhibitor of κB kinase (IKK)α activation and non-canonical NF-κB signaling. Non-canonical NF-κB signaling is terminated by feedback phosphorylation of NIK by IKKα that promotes NIK degradation; however, the mechanism of active NIK protein turnover remains unknown. To address this question, we established a strategy to precisely distinguish between basal degradation of newly synthesized endogenous NIK and induced active NIK in stimulated cells. Using this approach, we found that IKKα-mediated degradation of signal-induced activated NIK occurs through the proteasome. To determine whether cIAP1 or cIAP2 play a role in active NIK turnover, we utilized a Smac mimetic (GT13072), which promotes degradation of these E3 ubiquitin ligases. As expected, GT13072 stabilized NIK in resting cells. However, loss of the cIAPs did not inhibit proteasome-dependent turnover of signal-induced NIK showing that unlike the basal regulatory mechanism, active NIK turnover is independent of cIAP1 and cIAP2. Our results therefore establish that the negative feedback control of IKKα-mediated NIK turnover occurs via a novel proteasome-dependent and cIAP-independent mechanism. PMID:24942881

  14. Phase 1 study of twice-weekly ixazomib, an oral proteasome inhibitor, in relapsed/refractory multiple myeloma patients.

    PubMed

    Richardson, Paul G; Baz, Rachid; Wang, Michael; Jakubowiak, Andrzej J; Laubach, Jacob P; Harvey, R Donald; Talpaz, Moshe; Berg, Deborah; Liu, Guohui; Yu, Jiang; Gupta, Neeraj; Di Bacco, Alessandra; Hui, Ai-Min; Lonial, Sagar

    2014-08-14

    Ixazomib is the first investigational oral proteasome inhibitor to be studied clinically. In this phase 1 trial, 60 patients with relapsed/refractory multiple myeloma (median of 4 prior lines of therapy; bortezomib, lenalidomide, thalidomide, and carfilzomib/marizomib in 88%, 88%, 62%, and 5%, respectively) received single-agent ixazomib 0.24 to 2.23 mg/m(2) (days 1, 4, 8, 11; 21-day cycles). Two dose-limiting toxicities (grade 3 rash; grade 4 thrombocytopenia) occurred at 2.23 mg/m(2). The maximum tolerated dose was 2.0 mg/m(2), which 40 patients received in 4 expansion cohorts. Patients received a median of 4 cycles (range, 1-39); 18% received ≥12 cycles. Eighty-eight percent had drug-related adverse events, including nausea (42%), thrombocytopenia (42%), fatigue (40%), and rash (40%); drug-related grade ≥3 events included thrombocytopenia (37%) and neutropenia (17%). Grade 1/2 drug-related peripheral neuropathy occurred in 12% (no grade ≥3). Two patients died on the study (both considered unrelated to treatment). The terminal half-life of ixazomib was 3.3 to 7.4 days; plasma exposure increased proportionally with dose (0.48-2.23 mg/m(2)). Among 55 response-evaluable patients, 15% achieved partial response or better (76% stable disease or better). These findings have informed the subsequent clinical development of ixazomib in multiple myeloma. This trial was registered at www.clinicaltrials.gov as #NCT00932698. PMID:24920586

  15. Phase 1 study of twice-weekly ixazomib, an oral proteasome inhibitor, in relapsed/refractory multiple myeloma patients

    PubMed Central

    Baz, Rachid; Wang, Michael; Jakubowiak, Andrzej J.; Laubach, Jacob P.; Harvey, R. Donald; Talpaz, Moshe; Berg, Deborah; Liu, Guohui; Yu, Jiang; Gupta, Neeraj; Di Bacco, Alessandra; Hui, Ai-Min; Lonial, Sagar

    2014-01-01

    Ixazomib is the first investigational oral proteasome inhibitor to be studied clinically. In this phase 1 trial, 60 patients with relapsed/refractory multiple myeloma (median of 4 prior lines of therapy; bortezomib, lenalidomide, thalidomide, and carfilzomib/marizomib in 88%, 88%, 62%, and 5%, respectively) received single-agent ixazomib 0.24 to 2.23 mg/m2 (days 1, 4, 8, 11; 21-day cycles). Two dose-limiting toxicities (grade 3 rash; grade 4 thrombocytopenia) occurred at 2.23 mg/m2. The maximum tolerated dose was 2.0 mg/m2, which 40 patients received in 4 expansion cohorts. Patients received a median of 4 cycles (range, 1-39); 18% received ≥12 cycles. Eighty-eight percent had drug-related adverse events, including nausea (42%), thrombocytopenia (42%), fatigue (40%), and rash (40%); drug-related grade ≥3 events included thrombocytopenia (37%) and neutropenia (17%). Grade 1/2 drug-related peripheral neuropathy occurred in 12% (no grade ≥3). Two patients died on the study (both considered unrelated to treatment). The terminal half-life of ixazomib was 3.3 to 7.4 days; plasma exposure increased proportionally with dose (0.48-2.23 mg/m2). Among 55 response-evaluable patients, 15% achieved partial response or better (76% stable disease or better). These findings have informed the subsequent clinical development of ixazomib in multiple myeloma. This trial was registered at www.clinicaltrials.gov as #NCT00932698. PMID:24920586

  16. Switching from body surface area-based to fixed dosing for the investigational proteasome inhibitor ixazomib: a population pharmacokinetic analysis

    PubMed Central

    Gupta, Neeraj; Zhao, Yuan; Hui, Ai-Min; Esseltine, Dixie-Lee; Venkatakrishnan, Karthik

    2015-01-01

    Aims This population pharmacokinetic analysis of the investigational oral proteasome inhibitor ixazomib assessed the feasibility of switching from body surface area (BSA)-based to fixed dosing, and the impact of baseline covariates on ixazomib pharmacokinetics. Methods Data were pooled from 226 adult patients with multiple myeloma, lymphoma or solid tumours in four phase 1 studies, in which ixazomib dosing (oral/intravenous, once/twice weekly) was based on BSA. Population pharmacokinetic modelling was undertaken using nonmem version 7.2. Results Ixazomib pharmacokinetics were well described by a three compartment model with first order absorption and linear elimination. Ixazomib was absorbed rapidly (Ka 0.5 h−1), with dose- and time-independent pharmacokinetics. Estimated absolute bioavailability and clearance were 60% and 2 l h−1, respectively. Although a small effect of BSA (range 1.3–2.6 m2) was observed on the peripheral volume of distribution (V4), reducing the corresponding inter-individual variability by 12.9%, there was no relationship between BSA and ixazomib clearance (the parameter that dictates total systemic exposure following fixed dosing). Consistently, based on simulations (n = 1000), median AUCs (including interquartile range) were similar after BSA-based (2.23 mg m−2) and fixed (4 mg) oral dosing with no trend in simulated AUC vs. BSA for fixed dosing (P = 0.42). No other covariates, including creatinine clearance (22–213.7 ml min−1) and age (23–86 years), influenced ixazomib pharmacokinetics. Conclusions This analysis supports a switch from BSA-based to fixed dosing, without dose modification for mild/moderate renal impairment or age, in future adult studies of ixazomib, simplifying dosing guidance and clinical development. PMID:25377318

  17. Phase I Trial Using the Proteasome Inhibitor Bortezomib and Concurrent Chemoradiotherapy for Head-and-Neck Malignancies

    SciTech Connect

    Kubicek, Gregory J.; Axelrod, Rita S.; Machtay, Mitchell; Ahn, Peter H.; Anne, Pramila R.; Fogh, Shannon; Cognetti, David; Myers, Thomas J.; Curran, Walter J.; Dicker, Adam P.

    2012-07-15

    Purpose: Advanced head-and-neck cancer (HNC) remains a difficult disease to cure. Proteasome inhibitors such as bortezomib have the potential to improve survival over chemoradiotherapy alone. This Phase I dose-escalation study examined the potential of bortezomib in combination with cisplatin chemotherapy and concurrent radiation in the treatment of locally advanced and recurrent HNC. Methods and Materials: Eligible patients received cisplatin once weekly at 30 mg/m{sup 2} per week and bortezomib along with concurrent radiation. Bortezomib was given on Days 1, 4, 8, and 11 every 3 weeks, with an initial starting dose of 0.7 mg/m{sup 2} and escalation levels of 1.0 and 1.3 mg/m{sup 2}. Dose escalation was performed only after assessment to rule out any dose-limiting toxicity. Results: We enrolled 27 patients with HNC, including 17 patients with recurrent disease who had received prior irradiation. Patients received bortezomib dose levels of 0.7 mg/m{sup 2} (7 patients), 1.0 mg/m{sup 2} (10 patients), and 1.3 mg/m{sup 2} (10 patients). No Grade 5 toxicities, 3 Grade 4 toxicities (all hematologic and considered dose-limiting toxicities), and 39 Grade 3 toxicities (in 20 patients) were observed. With a median follow-up of 7.4 months, the overall median survival was 24.7 months (48.4 months for advanced HNC patients and 15.4 months for recurrent HNC patients). Conclusion: Bortezomib in combination with radiation therapy and cisplatin chemotherapy is safe in the treatment of HNC with a bortezomib maximum tolerated dose of 1.0 mg/m{sup 2} in patients previously treated for HNC and 1.3 mg/m{sup 2} in radiation-naive patients.

  18. Chemical and biological evaluation of dipeptidyl boronic acid proteasome inhibitors for use in prodrugs and pro-soft drugs targeting solid tumors.

    PubMed

    Milo, Lawrence J; Lai, Jack H; Wu, Wengen; Liu, Yuxin; Maw, Hlaing; Li, Youhua; Jin, Zhiping; Shu, Ying; Poplawski, Sarah E; Wu, Yong; Sanford, David G; Sudmeier, James L; Bachovchin, William W

    2011-07-14

    Bortezomib, a dipeptidyl boronic acid and potent inhibitor of the 26S proteasome, is remarkably effective against multiple myeloma (MM) but not against solid tumors. Dose-limiting adverse effects from "on target" inhibition of the proteasome in normal cells and tissues appear to be a key obstacle. Achieving efficacy against solid tumors therefore is likely to require making the inhibitor more selective for tumor tissue over normal tissues. The simplest strategy that might provide such tissue specificity would be to employ a tumor specific protease to release an inhibitor from a larger, noninhibitory structure. However, such release would necessarily generate an inhibitor with a free N-terminal amino group, raising a key question: Can short peptide boronic acids with N-terminal amino groups have the requisite properties to serve as warheads in prodrugs? Here we show that dipeptides of boroLeu, the smallest plausible candidates for the task, can indeed be sufficiently potent, cell-penetrating, cytotoxic, and stable to degradation by cellular peptidases to serve in this capacity. PMID:21634429

  19. Proteasome inhibitor PS-341 limits macrophage necroptosis by promoting cIAPs-mediated inhibition of RIP1 and RIP3 activation.

    PubMed

    Zhang, Yuchen; Cheng, Junjun; Zhang, Junmeng; Wu, Xiaofan; Chen, Fang; Ren, Xuejun; Wang, Yunlong; Li, Quan; Li, Yu

    2016-09-01

    Apoptotic and necrotic macrophages have long been known for their existence in atherosclerotic lesions. However, the mechanisms underlying the choice of their death pattern have not been fully elucidated. Here, we report the effects of PS-341, a potent and specific proteasome inhibitor, on the cell death of primary bone marrow-derived macrophages (BMDMs) in vitro. The results showed that PS-341 could not induce macrophage apoptosis or promote TNF-induced macrophage apoptosis, on the other hand, PS-341 could significantly inhibit macrophage necroptosis induced by TNF and pan-caspase inhibitor z-VAD treatment. Remarkably, high-dose of PS-341 showed similar inhibitory effects on macrophage necroptosis comparable to that of kinase inhibition of RIP1 through specific inhibitor Nec-1 or inhibition of RIP3 via specific genetical ablation. Furthermore, the degradation of cellular inhibitor of apoptosis proteins (cIAPs) was suppressed by PS-341, which could antagonize the activation of RIP1 kinase via post-translational mechanism. Further evidences demonstrated reduced levels of both RIP1 and RIP 3 upon PS-341 treatment, concomitantly, a more strong association of RIP1 with cIAPs and less with RIP3 was found following PS-341 treatment, these findings suggested that PS-341 may disrupt the formation of RIP1-RIP3 complex (necrosome) through stabilizing cIAPs. Collectively, our results indicated that the proteasome-mediated degradation of cIAPs could be inhibited by PS-341 and followed by limited RIP1 and RIP3 kinase activities, which were indispensable for necroptosis, thus eliciting a significant necroptosis rescue in BMDMs in vitro. Overall, our study has identified a new role of PS-341 in the cell death of BMDMs and provided a novel insight into the atherosclerotic inflammation caused by proteasome-mediated macrophage necroptosis. PMID:27363341

  20. A Bowman-Birk inhibitor induces apoptosis in human breast adenocarcinoma through mitochondrial impairment and oxidative damage following proteasome 20S inhibition.

    PubMed

    Mehdad, A; Brumana, G; Souza, A A; Barbosa, Jarg; Ventura, M M; de Freitas, S M

    2016-01-01

    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. PMID:27551492

  1. A Bowman–Birk inhibitor induces apoptosis in human breast adenocarcinoma through mitochondrial impairment and oxidative damage following proteasome 20S inhibition

    PubMed Central

    Mehdad, A; Brumana, G; Souza, AA; Barbosa, JARG; Ventura, MM; de Freitas, SM

    2016-01-01

    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. PMID:27551492

  2. The pan-HDAC inhibitor vorinostat potentiates the activity of the proteasome inhibitor carfilzomib in human DLBCL cells in vitro and in vivo.

    PubMed

    Dasmahapatra, Girija; Lembersky, Dmitry; Kramer, Lora; Fisher, Richard I; Friedberg, Jonathan; Dent, Paul; Grant, Steven

    2010-06-01

    Interactions between histone deacetylase inhibitors (HDACIs) and the novel proteasome inhibitor carfilzomib (CFZ) were investigated in GC- and activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL) cells. Coadministration of subtoxic or minimally toxic concentrations of CFZ) with marginally lethal concentrations of HDACIs (vorinostat, SNDX-275, or SBHA) synergistically increased mitochondrial injury, caspase activation, and apoptosis in both GC- and ABC-DLBCL cells. These events were associated with Jun NH2-terminal kinase (JNK) and p38MAPK activation, abrogation of HDACI-mediated nuclear factor-kappaB activation, AKT inactivation, Ku70 acetylation, and induction of gammaH2A.X. Genetic or pharmacologic JNK inhibition significantly diminished CFZ/vorinostat lethality. CFZ/vorinostat induced pronounced lethality in 3 primary DLBCL specimens but minimally affected normal CD34(+) hematopoietic cells. Bortezomib-resistant GC (SUDHL16) and ABC (OCI-LY10) cells exhibited partial cross-resistance to CFZ. However, CFZ/vorinostat dramatically induced resistant cell apoptosis, accompanied by increased JNK activation and gammaH2A.X expression. Finally, subeffective vorinostat doses markedly increased CFZ-mediated tumor growth suppression and apoptosis in a murine xenograft OCI-LY10 model. These findings indicate that HDACIs increase CFZ activity in GC- and ABC-DLBCL cells sensitive or resistant to bortezomib through a JNK-dependent mechanism in association with DNA damage and inhibition of nuclear factor-kappaB activation. Together, they support further investigation of strategies combining CFZ and HDACIs in DLBCL. PMID:20233973

  3. The pan-HDAC inhibitor vorinostat potentiates the activity of the proteasome inhibitor carfilzomib in human DLBCL cells in vitro and in vivo

    PubMed Central

    Dasmahapatra, Girija; Lembersky, Dmitry; Kramer, Lora; Fisher, Richard I.; Friedberg, Jonathan; Dent, Paul

    2010-01-01

    Interactions between histone deacetylase inhibitors (HDACIs) and the novel proteasome inhibitor carfilzomib (CFZ) were investigated in GC- and activated B-cell–like diffuse large B-cell lymphoma (ABC-DLBCL) cells. Coadministration of subtoxic or minimally toxic concentrations of CFZ) with marginally lethal concentrations of HDACIs (vorinostat, SNDX-275, or SBHA) synergistically increased mitochondrial injury, caspase activation, and apoptosis in both GC- and ABC-DLBCL cells. These events were associated with Jun NH2-terminal kinase (JNK) and p38MAPK activation, abrogation of HDACI-mediated nuclear factor-κB activation, AKT inactivation, Ku70 acetylation, and induction of γH2A.X. Genetic or pharmacologic JNK inhibition significantly diminished CFZ/vorinostat lethality. CFZ/vorinostat induced pronounced lethality in 3 primary DLBCL specimens but minimally affected normal CD34+ hematopoietic cells. Bortezomib-resistant GC (SUDHL16) and ABC (OCI-LY10) cells exhibited partial cross-resistance to CFZ. However, CFZ/vorinostat dramatically induced resistant cell apoptosis, accompanied by increased JNK activation and γH2A.X expression. Finally, subeffective vorinostat doses markedly increased CFZ-mediated tumor growth suppression and apoptosis in a murine xenograft OCI-LY10 model. These findings indicate that HDACIs increase CFZ activity in GC- and ABC-DLBCL cells sensitive or resistant to bortezomib through a JNK-dependent mechanism in association with DNA damage and inhibition of nuclear factor-κB activation. Together, they support further investigation of strategies combining CFZ and HDACIs in DLBCL. PMID:20233973

  4. IN VITRO AND IN VIVO INTERACTIONS BETWEEN THE HDAC6 INHIBITOR RICOLINOSTAT (ACY1215) AND THE IRREVERSIBLE PROTEASOME INHIBITOR CARFILZOMIB IN NON-HODGKIN’S LYMPHOMA CELLS

    PubMed Central

    Dasmahapatra, Girija; Patel, Hiral; Friedberg, Johnathan; Quayle, Steven N; Jones, Simon S; Grant, Steven

    2014-01-01

    Interactions between the HDAC6 inhibitor ricolinostat (ACY1215) and the irreversible proteasome inhibitor Carfilzomib (CFZ) were examined in non-Hodgkin’s lymphoma models, including diffuse large B-cell (DLBCL), mantle cell (MCL) and double-hit lymphoma cells. Marked in vitro synergism was observed in multiple cell types associated with activation of cellular stress pathways (e.g., JNK1/2, ERK1/2, and p38) accompanied by increases in DNA damage (γH2A.X), G2M arrest, and the pronounced induction of mitochondrial injury and apoptosis. Combination treatment with CFZ and ricolinostat increased reactive oxygen species (ROS), while the antioxidant TBAP attenuated DNA damage, JNK activation, and cell death. Similar interactions occurred in bortezomib-resistant and double-hit DLBCL, MCL, and primary DLBCL cells, but not in normal CD34+ cells. However, ricolinostat did not potentiate inhibition of chymotryptic activity by CFZ. shRNA knock-down of JNK1 (but not MEK1/2), or pharmacologic inhibition of p38, significantly reduced CFZ/ricolinostat lethality, indicating a functional contribution of these stress pathways to apoptosis. Combined exposure to CFZ and ricolinostat also markedly down-regulated the cargo-loading protein HR23B. Moreover, HR23B knock-down significantly increased CFZ- and ricolinostat-mediated lethality, suggesting a role for this event in cell death. Finally, combined in vivo treatment with CFZ and ricolinostat was well tolerated and significantly suppressed tumor growth and increased survival in an MCL xenograft model. Collectively, these findings indicate that CFZ and ricolinostat interact synergistically in NHL cells through multiple stress-related mechanisms, and suggest that this strategy warrants further consideration in NHL. PMID:25239935

  5. NITRIC OXIDE-DEPENDENT PROTEASOMAL DEGRADATION OF CYTOCHROME P450 2B PROTEINS*

    PubMed Central

    Lee, Choon-Myung; Kim, Bong-Yoon; Li, Lian; Morgan, Edward T.

    2007-01-01

    Exposure to inflammatory agents or cytokines causes the suppression of cytochrome P450 (CYP) enzyme activities and expression in liver and primary hepatocyte cultures. We showed previously that phenobarbital-induced CYP2B protein is down-regulated in primary cultures of rat hepatocytes following exposure to bacterial endotoxin (LPS) in a nitric oxide (NO)-dependent manner. In the present study, we found that CYP2B proteins in primary rat hepatocyte cultures were suppressed more than 60% after 6h treatment with interleukin-1β (IL-1). This effect was NO-dependent, and treatment of cells with the NO-donors (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino] diazen-1-ium-1,2-diolate (NOC-18), S-nitrosoglutathione (GSNO), and S-nitroso, N-acetylpenicillamine (SNAP) also suppressed CYP2B proteins. However, the down-regulation by IL-1 was insensitive to inhibition of cGMP-dependent protein kinases. The down-regulation by IL-1 or NO donors was abolished by treatments with the proteasome inhibitors MG132 and lactacystin that did not affect NO production. The calpain inhibitor E64-d or the lysosomal protease inhibitors NH4Cl and chloroquine did not attenuate the down-regulation of CYP2B by IL-1. Treatment of HeLa cells expressing c-myc-tagged CYP2B1 with NOC-18 down-regulated its expression and enhanced its ubiquitination. Treatment of rat liver microsomes with GSNO caused S-nitrosylation of CYP2B protein, and enhanced the ubiquitination pattern of CYP2B compared to unmodified CYP2B in an in vitro ubiquitination assay. These data are consistent with the hypothesis that NO-dependent CYP2B ubiquitination and proteasomal degradation are dependent on protein modification by reactive nitrogen species. PMID:17993647

  6. MDM2 promotes p21waf1/cip1 proteasomal turnover independently of ubiquitylation.

    PubMed

    Jin, Yetao; Lee, Hunjoo; Zeng, Shelya X; Dai, Mu-Shui; Lu, Hua

    2003-12-01

    The CDK inhibitor p21waf1/cip1 is degraded by a ubiquitin-independent proteolytic pathway. Here, we show that MDM2 mediates this degradation process. Overexpression of wild-type or ring finger-deleted, but not nuclear localization signal (NLS)-deleted, MDM2 decreased p21waf1/cip1 levels without ubiquitylating this protein and affecting its mRNA level in p53(-/-) cells. This decrease was reversed by the proteasome inhibitors MG132 and lactacystin, by p19(arf), and by small interfering RNA (siRNA) against MDM2. p21waf1/cip1 bound to MDM2 in vitro and in cells. The p21waf1/cip1-binding-defective mutant of MDM2 was unable to degrade p21waf1/cip1. MDM2 shortened the half-life of both exogenous and endogenous p21waf1/cip1 by 50% and led to the degradation of its lysine-free mutant. Consequently, MDM2 suppressed p21waf1/cip1-induced cell growth arrest of human p53(-/-) and p53(-/-)/Rb(-/-)cells. These results demonstrate that MDM2 directly inhibits p21waf1/cip1 function by reducing p21waf1/cip1 stability in a ubiquitin-independent fashion. PMID:14633995

  7. Mechanism of Action of Bortezomib and the New Proteasome Inhibitors on Myeloma Cells and the Bone Microenvironment: Impact on Myeloma-Induced Alterations of Bone Remodeling

    PubMed Central

    Accardi, Fabrizio; Toscani, Denise; Bolzoni, Marina; Dalla Palma, Benedetta; Aversa, Franco; Giuliani, Nicola

    2015-01-01

    Multiple myeloma (MM) is characterized by a high capacity to induce alterations in the bone remodeling process. The increase in osteoclastogenesis and the suppression of osteoblast formation are both involved in the pathophysiology of the bone lesions in MM. The proteasome inhibitor (PI) bortezomib is the first drug designed and approved for the treatment of MM patients by targeting the proteasome. However, recently novel PIs have been developed to overcome bortezomib resistance. Interestingly, several preclinical data indicate that the proteasome complex is involved in both osteoclast and osteoblast formation. It is also evident that bortezomib either inhibits osteoclast differentiation induced by the receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) or stimulates the osteoblast differentiation. Similarly, the new PIs including carfilzomib and ixazomib can inhibit bone resorption and stimulate the osteoblast differentiation. In a clinical setting, PIs restore the abnormal bone remodeling by normalizing the levels of bone turnover markers. In addition, a bone anabolic effect was described in responding MM patients treated with PIs, as demonstrated by the increase in the osteoblast number. This review summarizes the preclinical and clinical evidence on the effects of bortezomib and other new PIs on myeloma bone disease. PMID:26579531

  8. Mechanism of Action of Bortezomib and the New Proteasome Inhibitors on Myeloma Cells and the Bone Microenvironment: Impact on Myeloma-Induced Alterations of Bone Remodeling.

    PubMed

    Accardi, Fabrizio; Toscani, Denise; Bolzoni, Marina; Dalla Palma, Benedetta; Aversa, Franco; Giuliani, Nicola

    2015-01-01

    Multiple myeloma (MM) is characterized by a high capacity to induce alterations in the bone remodeling process. The increase in osteoclastogenesis and the suppression of osteoblast formation are both involved in the pathophysiology of the bone lesions in MM. The proteasome inhibitor (PI) bortezomib is the first drug designed and approved for the treatment of MM patients by targeting the proteasome. However, recently novel PIs have been developed to overcome bortezomib resistance. Interestingly, several preclinical data indicate that the proteasome complex is involved in both osteoclast and osteoblast formation. It is also evident that bortezomib either inhibits osteoclast differentiation induced by the receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) or stimulates the osteoblast differentiation. Similarly, the new PIs including carfilzomib and ixazomib can inhibit bone resorption and stimulate the osteoblast differentiation. In a clinical setting, PIs restore the abnormal bone remodeling by normalizing the levels of bone turnover markers. In addition, a bone anabolic effect was described in responding MM patients treated with PIs, as demonstrated by the increase in the osteoblast number. This review summarizes the preclinical and clinical evidence on the effects of bortezomib and other new PIs on myeloma bone disease. PMID:26579531

  9. Crystal structure of N-{N-[N-acetyl-(S)-leucyl]-(S)-leucyl}norleucinal (ALLN), an inhibitor of proteasome

    SciTech Connect

    Czerwinski, Andrzej; Basava, Channa; Dauter, Miroslawa; Dauter, Zbigniew

    2015-03-01

    The title compound, C20H37N3O4, also known by the acronym ALLN, is a tripeptidic inhibitor of the proteolytic activity of the proteasomes, enzyme complexes implicated in several neurodegenerative diseases and other disorders, including cancer. Thus, the crystal structure of ALLN, solved from synchrotron radiation diffraction data, revealed the molecules in extended conformation of the backbone and engaging all peptide N and O atoms in intermolecular hydrogen bonds forming an infinite antiparallel β-sheet.

  10. The proteasome deubiquitinase inhibitor VLX1570 shows selectivity for ubiquitin-specific protease-14 and induces apoptosis of multiple myeloma cells.

    PubMed

    Wang, Xin; Mazurkiewicz, Magdalena; Hillert, Ellin-Kristina; Olofsson, Maria Hägg; Pierrou, Stefan; Hillertz, Per; Gullbo, Joachim; Selvaraju, Karthik; Paulus, Aneel; Akhtar, Sharoon; Bossler, Felicitas; Khan, Asher Chanan; Linder, Stig; D'Arcy, Padraig

    2016-01-01

    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. PMID:27264969

  11. Proteasome inhibitor MG132 inhibits the proliferation and promotes the cisplatin-induced apoptosis of human esophageal squamous cell carcinoma cells

    PubMed Central

    DANG, LIFENG; WEN, FENGBIAO; YANG, YANG; LIU, DONGLEI; WU, KAI; QI, YU; LI, XIANGNAN; ZHAO, JIA; ZHU, DENGYAN; ZHANG, CHUNYANG; ZHAO, SONG

    2014-01-01

    Comprehensive treatment based on chemotherapy is regarded as the first-line treatment for patients with unresectable or metastatic esophageal squamous cell carcinoma (ESCC). However, chemoresistance is common among patients with ESCC. Therefore, there is a need to explore new therapeutic strategies or adjuvant drugs. One promising possibility is to use dietary agents that can increase tumor cell sensitivity to drugs. In this study, we initially investigated the antitumor activity of proteasome inhibitor MG132 in vitro and in vivo. Effects of MG132 on the enhancment of the anticancer functions of cisplatin were then investigated in human esophageal cancer EC9706 cells in relation to apoptosis and cell signaling events. Exposure of cells to MG132 resulted in a marked decrease in cell viability in a dose- and time-dependent manner. Administration of MG132 markedly inhibited tumor growth in the EC9706 xenograft model. MG132 significantly enhanced cisplatin-induced apoptosis in association with the activation of caspase-3 and -8. These events were accompanied by the downregulation of NF-κB, which plays a key role in cell apoptosis. Taken together, these findings demonstrate a novel mechanism by which proteasome inhibitor MG132 potentiates cisplatin-induced apoptosis in human ESCC and inhibitory activity of tumor growth of the EC9706 xenograft model. PMID:24584782

  12. Combination treatment with proteasome inhibitors and antiestrogens has a synergistic effect mediated by p21WAF1 in estrogen receptor-positive breast cancer.

    PubMed

    Maynadier, Marie; Basile, Ilaria; Gallud, Audrey; Gary-Bobo, Magali; Garcia, Marcel

    2016-08-01

    Although antiestrogens significantly improve the survival of patients with ER-positive breast cancer, therapeutic resistance remains a major limitation. The combinatorial use of antiestrogen with other therapies was proposed to increase their efficiency and more importantly, to prevent or delay the resistance phenomenon. In the present study, we addressed their combined effects with proteasome inhibitors (PIs). The effects of antiestrogens (hydroxyl-tamoxifen, raloxifen and fulvestrant) currently used in endocrine therapy were tested in combination with PIs, bortezomib or MG132, on the growth of three ER-positive breast cancer cell lines and in two cellular models of acquired antiestrogen resistance. When compared to single treatments, these combined treatments were significantly more effective in preventing the growth of the cell lines. The regulation of key cell cycle proteins, the cyclin-dependent kinase inhibitors, p21WAF1 and p27KIP1, were also studied. Bortezomib and MG132 drastically increased p21WAF1 expression through elevation of its mRNA concentration. Notably, p27KIP1 regulation was quite different from that of p21WAF1. Furthermore, the effect of bortezomib in combination with antiestrogen was evaluated on antiestrogen-resistant cell lines. The growth of two antiestrogen-resistant cell lines appeared responsive to proteasome inhibition and was strongly decreased by a combined therapy with an antiestrogen. Collectively, these findings provide new perspectives for the use of PIs in combination with endocrine therapies for breast cancer and possibly to overcome acquired hormonal resistance. PMID:27373750

  13. The proteasome deubiquitinase inhibitor VLX1570 shows selectivity for ubiquitin-specific protease-14 and induces apoptosis of multiple myeloma cells

    PubMed Central

    Wang, Xin; Mazurkiewicz, Magdalena; Hillert, Ellin-Kristina; Olofsson, Maria Hägg; Pierrou, Stefan; Hillertz, Per; Gullbo, Joachim; Selvaraju, Karthik; Paulus, Aneel; Akhtar, Sharoon; Bossler, Felicitas; Khan, Asher Chanan; Linder, Stig; D’Arcy, Padraig

    2016-01-01

    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. PMID:27264969

  14. E11/Podoplanin Protein Stabilization Through Inhibition of the Proteasome Promotes Osteocyte Differentiation in Murine in Vitro Models

    PubMed Central

    Prideaux, Matt; Allen, Steve; Buttle, David J.; Pitsillides, Andrew A.; Farquharson, Colin

    2015-01-01

    The transmembrane glycoprotein E11 is considered critical in early osteoblast–osteocyte transitions (osteocytogenesis), however its function and regulatory mechanisms are still unknown. Using the late osteoblast MLO‐A5 cell line we reveal increased E11 protein/mRNA expression (P < 0.001) concomitant with extensive osteocyte dendrite formation and matrix mineralization (P < 0.001). Transfection with E11 significantly increased mRNA levels (P < 0.001), but immunoblotting failed to detect any correlative increases in E11 protein levels, suggestive of post‐translational degradation. We found that exogenous treatment of MLO‐A5 and osteocytic IDG‐SW3 cells with 10 μM ALLN (calpain and proteasome inhibitor) stabilized E11 protein levels and induced a profound increase in osteocytic dendrite formation (P < 0.001). Treatment with other calpain inhibitors failed to promote similar osteocytogenic changes, suggesting that these effects of ALLN rely upon its proteasome inhibitor actions. Accordingly we found that proteasome‐selective inhibitors (MG132/lactacystin/ Bortezomib/Withaferin‐A) produced similar dose‐dependent increases in E11 protein levels in MLO‐A5 and primary osteoblast cells. This proteasomal targeting was confirmed by immunoprecipitation of ubiquitinylated proteins, which included E11, and by increased levels of ubiquitinylated E11 protein upon addition of the proteasome inhibitors MG132/Bortezomib. Activation of RhoA, the small GTPase, was found to be increased concomitant with the peak in E11 levels and its downstream signaling was also observed to promote MLO‐A5 cell dendrite formation. Our data indicate that a mechanism reliant upon blockade of proteasome‐mediated E11 destabilization contributes to osteocytogenesis and that this may involve downstream targeting of RhoA. This work adds to our mechanistic understanding of the factors regulating bone homeostasis, which may lead to future therapeutic approaches. J. Cell

  15. The long N-terminus of the human monocarboxylate transporter 8 is a target of ubiquitin-dependent proteasomal degradation which regulates protein expression and oligomerization capacity.

    PubMed

    Zwanziger, Denise; Schmidt, Mathias; Fischer, Jana; Kleinau, Gunnar; Braun, Doreen; Schweizer, Ulrich; Moeller, Lars Christian; Biebermann, Heike; Fuehrer, Dagmar

    2016-10-15

    Monocarboxylate transporter 8 (MCT8) equilibrates thyroid hormones between the extra- and the intracellular sides. MCT8 exists either with a short or a long N-terminus, but potential functional differences between both variants are yet not known. We, therefore, generated MCT8 constructs which are different in N-terminal length: MCT8(1-613), MCT8(25-613), MCT8(49-613) and MCT8(75-613). The M75G substitution prevents translation of MCT8(75-613) and ensures expression of full-length MCT8 protein. The K56G substitution was made to prevent ubiquitinylation. Cell-surface expression, localization and proteasomal degradation were investigated using C-terminally GFP-tagged MCT8 constructs (HEK293 and MDCK1 cells) and oligomerization capacity was determined using N-terminally HA- and C-terminally FLAG-tagged MCT8 constructs (COS7 cells). MCT8(1-613)-GFP showed a lower protein expression than the shorter MCT8(75-613)-GFP protein. The proteasome inhibitor lactacystin increased MCT8(1-613)-GFP protein amount, suggesting proteasomal degradation of MCT8 with the long N-terminus. Ubiquitin conjugation of MCT8(1-613)-GFP was found by immuno-precipitation. A diminished ubiquitin conjugation caused by K56G substitution resulted in increased MCT8(1-613)-GFP protein expression. Sandwich ELISA was performed to investigate if the bands at higher molecular weight observed in Western blot analysis are due to MCT8 oligomerization, which was indeed shown. Our data imply a role of the long N-terminus of MCT8 as target of ubiquitin-dependent proteasomal degradation affecting MCT8 amount and subsequently oligomerization capacity. PMID:27222294

  16. The Proteasome Inhibitor Carfilzomib Functions Independently of p53 To Induce Cytotoxicity and an Atypical NF-κB Response in Chronic Lymphocytic Leukemia Cells

    PubMed Central

    Gupta, Sneha V.; Hertlein, Erin; Lu, Yanhui; Sass, Ellen J.; Lapalombella, Rosa; Chen, Timothy L.; Davis, Melanie E.; Woyach, Jennifer A.; Lehman, Amy; Jarjoura, David; Byrd, John C.; Lucas, David M.

    2013-01-01

    Purpose The proteasome consists of chymotrypsin-like (CT-L), trypsin-like, and caspase-like subunits that cleave substrates preferentially by amino acid sequence. Proteasomes mediate degradation of regulatory proteins of the p53, Bcl-2 and nuclear factor-κB (NF-κB) families that are aberrantly active in chronic lymphocytic leukemia (CLL). CLL remains an incurable disease, and new treatments are especially needed in the relapsed/refractory setting. We therefore investigated the effects of the proteasome inhibitor carfilzomib (CFZ) in CLL cells. Experimental Design Tumor cells from CLL patients were assayed in vitro using immunoblotting, real-time polymerase chain reaction and electrophoretic mobility shift assays. Additionally, a p53 dominant-negative construct was generated in a human B-cell line. Results Unlike bortezomib, CFZ potently induces apoptosis in CLL patient cells in the presence of human serum. CLL cells have significantly lower basal CT-L activity compared to normal B and T cells, although activity is inhibited similarly in T cells vs. CLL. and the cytotoxicity of CFZ correlates with baseline CT-L activity. Co-culture of CLL cells on stroma protected from CFZ-mediated cytotoxicity; however, PI3K inhibition significantly diminished this stromal protection. CFZ-mediated cytotoxicity in leukemic B-cells is caspase-dependent and occurs irrespective of p53 status. In CLL cells, CFZ promotes atypical activation of NF-κB evidenced by loss of cytoplasmic IkBα, phosphorylation of IκBα and increased p50/p65 DNA binding, without subsequent increases in canonical NF-κB target gene transcription. Conclusions Together, these data provide new mechanistic insights into the activity of CFZ in CLL and support Phase I investigation of CFZ in this disease. PMID:23515408

  17. Characterizing the Dynamics of Proteasome Complexes by Proteomics Approaches

    PubMed Central

    Kaake, Robyn M.; Kao, Athit; Yu, Clinton

    2014-01-01

    Abstract Significance: The proteasome is the degradation machine of the ubiquitin-proteasome system, which is critical in controlling many essential biological processes. Aberrant regulation of proteasome-dependent protein degradation can lead to various human diseases, and general proteasome inhibitors have shown efficacy for cancer treatments. Though clinically effective, current proteasome inhibitors have detrimental side effects and, thus, better therapeutic strategies targeting proteasomes are needed. Therefore, a comprehensive characterization of proteasome complexes will provide the molecular details that are essential for developing new and improved drugs. Recent Advances: New mass spectrometry (MS)-based proteomics approaches have been developed to study protein interaction networks and structural topologies of proteasome complexes. The results have helped define the dynamic proteomes of proteasome complexes, thus providing new insights into the mechanisms underlying proteasome function and regulation. Critical Issues: The proteasome exists as heterogeneous populations in tissues/cells, and its proteome is highly dynamic and complex. In addition, proteasome complexes are regulated by various mechanisms under different physiological conditions. Consequently, complete proteomic profiling of proteasome complexes remains a major challenge for the field. Future Directions: We expect that proteomic methodologies enabling full characterization of proteasome complexes will continue to evolve. Further advances in MS instrumentation and protein separation techniques will be needed to facilitate the detailed proteomic analysis of low-abundance components and subpopulations of proteasome complexes. The results will help us understand proteasome biology as well as provide new therapeutic targets for disease diagnostics and treatment. Antioxid. Redox Signal. 21, 2444–2456. PMID:24423446

  18. Reduced O glycosylation of Sp1 is associated with increased proteasome susceptibility.

    PubMed Central

    Han, I; Kudlow, J E

    1997-01-01

    Sp1 is a ubiquitously expressed transcription factor that is particularly important for the regulation of TATA-less genes that encode housekeeping proteins. Most growth factors and receptors are also encoded by such genes. Sp1 is multiply O glycosylated by covalent linkage of the monosaccharide N-acetylglucosamine (O-GlcNAc) to serine and threonine residues. Based on an earlier observation that growth factor gene transcription can be regulated by glucose and glucosamine in vascular smooth muscle cells, we determined whether Sp1 glycosylation could be regulated and if this modification altered Sp1 function. We found that Sp1 becomes hyperglycosylated when cells are exposed to 5 mM glucosamine, whereas under glucose starvation, stimulation with cyclic AMP (cAMP) results in nearly complete deglycosylation of this protein. Correlating with this hypoglycosylated state, Sp1 is rapidly proteolytically degraded by an enzyme(s) that can be inhibited by specific proteasome inhibitors, lactacystin and LLnL. Treatment of cells with glucose or glucosamine protects Sp1 from cAMP-mediated degradation, whereas blockade of glucosamine synthesis abrogates glucose but not glucosamine protection. This effect on Sp1 is specific, in that the Stat-3 and E2F transcription factors did not undergo degradation under these conditions. The O-GlcNAc modification of Sp1 may play a role as a nutritional checkpoint. In the absence of adequate nutrition, Sp1 becomes hypoglycosylated and thereby subject to proteasome degradation. This process could potentially result in reduced general transcription, thereby conserving nutrients. PMID:9111324

  19. Dendritic Glycopolymer as Drug Delivery System for Proteasome Inhibitor Bortezomib in a Calcium Phosphate Bone Cement: First Steps Toward a Local Therapy of Osteolytic Bone Lesions.

    PubMed

    Striegler, Christin; Schumacher, Matthias; Effenberg, Christiane; Müller, Martin; Seckinger, Anja; Schnettler, Reinhard; Voit, Brigitte; Hose, Dirk; Gelinsky, Michael; Appelhans, Dietmar

    2015-09-01

    Establishment of drug delivery system (DDS) in bone substitute materials for local treatment of bone defects still requires ambitious solutions for a retarded drug release. We present two novel DDS, a weakly cationic dendritic glycopolymer and a cationic polyelectrolyte complex, composed of dendritic glycopolymer and cellulose sulfate, for the proteasome inhibitor bortezomib. Both DDS are able to induce short-term retarded release of bortezomib from calcium phosphate bone cement in comparison to a burst-release of the drug from bone cement alone. Different release parameters have been evaluated to get a first insight into the release mechanism from bone cements. In addition, biocompatibility of the calcium phosphate cement, modified with the new DDS was investigated using human mesenchymal stromal cells. PMID:26018141

  20. MG132, a proteasome inhibitor, enhances LDL uptake in HepG2 cells in vitro by regulating LDLR and PCSK9 expression

    PubMed Central

    Yan, Hong; Ma, Yan-ling; Gui, Yu-zhou; Wang, Shu-mei; Wang, Xin-bo; Gao, Fei; Wang, Yi-ping

    2014-01-01

    Aim: Expression of liver low-density lipoprotein receptor (LDLR), a determinant regulator in cholesterol homeostasis, is tightly controlled at multiple levels. The aim of this study was to examine whether proteasome inhibition could affect LDLR expression and LDL uptake in liver cells in vitro. Methods: HepG2 cells were examined. Real-time PCR and Western blot analysis were used to determine the mRNA and protein levels, respectively. DiI-LDL uptake assay was used to quantify the LDLR function. Luciferase assay system was used to detect the activity of proprotein convertase subtilisin/kexin type 9 (PCSK9, a major protein mediating LDLR degradation) promoter. Specific siRNAs were used to verify the involvement of PCSK9. Results: Treatment of HepG2 cells with the specific proteasome inhibitor MG132 (0.03–3 μmol/L) dose-dependently increased LDLR mRNA and protein levels, as well as LDL uptake. Short-term treatment with MG132 (0.3 μmol/L, up to 8 h) significantly increased both LDLR mRNA and protein levels in HepG2 cells, which was blocked by the specific PKC inhibitors GF 109203X, Gö 6983 or staurosporine. In contrast, a longer treatment with MG132 (0.3 μmol/L, 24 h) did not change LDLR mRNA, but markedly increased LDLR protein by reducing PCSK9-mediated lysosome LDLR degradation. Furthermore, MG132 time-dependently suppressed PCSK9 expression in the HepG2 cells through a SREBP-1c related pathway. Combined treatment with MG132 (0.3 μmol/L) and pravastatin (5 μmol/L) strongly promoted LDLR expression and LDL uptake in HepG2 cells, and blocked the upregulation of PCSK9 caused by pravastatin alone. Conclusion: Inhibition of proteasome by MG132 in HepG2 cells plays dual roles in LDLR and PCSK9 expression, and exerts a beneficial effect on cholesterol homeostasis. PMID:25042549

  1. Proteasome inhibitor MG-132 enhances histone deacetylase inhibitor SAHA-induced cell death of chronic myeloid leukemia cells by an ROS-mediated mechanism and downregulation of the Bcr-Abl fusion protein

    PubMed Central

    ZHOU, WENJING; ZHU, WEIWEI; MA, LIYA; XIAO, FENG; QIAN, WENBIN

    2015-01-01

    Recently, there has been progress in the treatment of chronic myeloid leukemia (CML). However, novel therapeutic strategies are required in order to address the emerging problem of imatinib resistance. Histone deacetylase inhibitors (HDACi) and proteasome inhibitors are promising alternatives, and may be amenable to integration with current therapeutic approaches. However, the mechanisms underlying the interaction between these two agents remain unclear. The present study assessed the cytotoxic effect of the HDACi, suberoylanilide hydroxamic acid (SAHA), in combination with the proteasome inhibitor, MG-132, in imatinib-sensitive K562 and imatinib-resistant K562G cells, and investigated the mechanism underlying this effect. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method and protein expression levels were determined by western blotting. Reactive oxygen species (ROS) generation levels were observed under a fluorescence microscope The results indicated that SAHA and MG-132 act in a synergistic manner to induce cell death in K562 and K562G cells. This effect was associated with Bcr-Abl downregulation and the production of ROS. Notably, the ROS scavenger, N-acetyl-L-cysteine, almost fully reversed the cell death and Bcr-Abl downregulation that was induced by the combination of SAHA and MG-132. By contrast, the pan-caspase inhibitor, z-VAD-fmk, only partially reversed the cell death induced by these two drugs in CML cells. These results indicated that increased intracellular ROS levels are important in the induction of cell death and the downregulation of Bcr-Abl. In conclusion, the present results suggested that combined SAHA and MG-132 may be a promising treatment for CML. PMID:26722260

  2. Steroidogenic acute regulatory protein gene expression, steroid-hormone secretion and proliferative activity of adrenocortical cells in the presence of proteasome inhibitors: in vivo studies on the regenerating rat adrenal cortex.

    PubMed

    Rucinski, Marcin; Tortorella, Cinzia; Ziolkowska, Agnieszka; Nowak, Magdalena; Nussdorfer, Gastone G; Malendowicz, Ludwik K

    2008-05-01

    Previous studies have shown that proteasome inhibitors promote the accumulation of steroidogenic acute regulatory protein (StAR) in cultured rat adrenocortical cells. Unexpectedly, this response was associated with a moderate lowering in the corticosterone secretion and proliferation rate of cultured cells. Hence, we studied the effects of proteasome inhibitors MG115 and MG132 on the secretion and proliferative activity of the regenerating adrenal cortex in rats 5 days after surgery. Animals were given two subcutaneous injections of 0.15 or 1.5 nmol/100 g of inhibitors 24 and 12 h before decapitation. Real-time PCR and Western blotting showed that StAR expression, both mRNA and protein, was markedly lower in regenerating adrenals than in the intact gland of sham-operated rats. Neither MG115 nor MG132 affected StAR expression in regenerating gland. Inhibitors induced a slight decrease in the plasma concentrations of aldosterone and corticosterone, but did not significantly alter metaphase index of the regenerating adrenal cortex. Our findings provide the first evidence that down-regulation of StAR occurs during the early stages of adrenal regeneration. Moreover, this suggests that the steroidogenic pathway is more sensitive to proteasome inhibitors than that regulating proliferative activity of regenerating adrenal cortex in the rat. PMID:18425351

  3. Structural Biology of the Proteasome

    PubMed Central

    Kish-Trier, Erik

    2016-01-01

    The proteasome refers to a collection of complexes centered on the 20S proteasome core particle, a complex of 28 subunits that houses proteolytic sites in its hollow interior. Proteasomes are found in eukaryotes, archaea, and some eubacteria, and their activity is critical for many cellular pathways. Important advances include inhibitor binding studies and the structure of the immunoproteasome, whose specificity is altered by incorporation of inducible catalytic subunits. The inherent repression of the 20S CP is relieved by the ATP-independent activators, 11S and Blm10/PA200, whose structures reveal principles of proteasome mechanism. The structure of the ATP-dependent 19S regulatory particle, which mediates degradation of polyubiquitylated proteins, is being revealed by a combination of crystal or NMR structures of individual subunits and electron microscopy reconstruction of the intact complex. Other recent structural advances inform about mechanisms of assembly and the role of conformational changes in the functional cycle. PMID:23414347

  4. Thiostrepton interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates

    PubMed Central

    Sandu, Cristinel; Chandramouli, Nagaranjan; Glickman, Joseph Fraser; Molina, Henrik; Kuo, Chueh-Ling; Kukushkin, Nikolay; Goldberg, Alfred L; Steller, Hermann

    2015-01-01

    Here, we report a novel mechanism of proteasome inhibition mediated by Thiostrepton (Thsp), which interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates. We identified Thsp in a cell-based high-throughput screen using a fluorescent reporter sensitive to degradation by the ubiquitin–proteasome pathway. Thiostrepton behaves as a proteasome inhibitor in several paradigms, including cell-based reporters, detection of global ubiquitination status, and proteasome-mediated labile protein degradation. In vitro, Thsp does not block the chymotrypsin activity of the 26S proteasome. In a cell-based IκBα degradation assay, Thsp is a slow inhibitor and 4 hrs of treatment achieves the same effects as MG-132 at 30 min. We show that Thsp forms covalent adducts with proteins in human cells and demonstrate their nature by mass spectrometry. Furthermore, the ability of Thsp to interact covalently with the cysteine residues is essential for its proteasome inhibitory function. We further show that a Thsp modified peptide cannot be degraded by proteasomes in vitro. Importantly, we demonstrate that Thsp binds covalently to Rpt subunits of the 19S regulatory particle and forms bridges with a proteasome substrate. Taken together, our results uncover an important role of Thsp in 19S proteasome inhibition. PMID:26033448

  5. Multi-output Model with Box-Jenkins Operators of Quadratic Indices for Prediction of Malaria and Cancer Inhibitors Targeting Ubiquitin- Proteasome Pathway (UPP) Proteins.

    PubMed

    Casañola-Martin, Gerardo M; Le-Thi-Thu, Huong; Pérez-Giménez, Facundo; Marrero-Ponce, Yovani; Merino-Sanjuán, Matilde; Abad, Concepción; González-Díaz, Humberto

    2016-01-01

    The ubiquitin-proteasome pathway (UPP) is the primary degradation system of short-lived regulatory proteins. Cellular processes such as the cell cycle, signal transduction, gene expression, DNA repair and apoptosis are regulated by this UPP and dysfunctions in this system have important implications in the development of cancer, neurodegenerative, cardiac and other human pathologies. UPP seems also to be very important in the function of eukaryote cells of the human parasites like Plasmodium falciparum, the causal agent of the neglected disease Malaria. Hence, the UPP could be considered as an attractive target for the development of compounds with Anti-Malarial or Anti-cancer properties. Recent online databases like ChEMBL contains a larger quantity of information in terms of pharmacological assay protocols and compounds tested as UPP inhibitors under many different conditions. This large amount of data give new openings for the computer-aided identification of UPP inhibitors, but the intrinsic data diversity is an obstacle for the development of successful classifiers. To solve this problem here we used the Bob-Jenkins moving average operators and the atom-based quadratic molecular indices calculated with the software TOMOCOMD-CARDD (TC) to develop a quantitative model for the prediction of the multiple outputs in this complex dataset. Our multi-target model can predict results for drugs against 22 molecular or cellular targets of different organisms with accuracies above 70% in both training and validation sets. PMID:26427384

  6. Mifepristone increases mRNA translation rate, triggers the unfolded protein response, increases autophagic flux, and kills ovarian cancer cells in combination with proteasome or lysosome inhibitors.

    PubMed

    Zhang, Lei; Hapon, Maria B; Goyeneche, Alicia A; Srinivasan, Rekha; Gamarra-Luques, Carlos D; Callegari, Eduardo A; Drappeau, Donis D; Terpstra, Erin J; Pan, Bo; Knapp, Jennifer R; Chien, Jeremy; Wang, Xuejun; Eyster, Kathleen M; Telleria, Carlos M

    2016-08-01

    The synthetic steroid mifepristone blocks the growth of ovarian cancer cells, yet the mechanism driving such effect is not entirely understood. Unbiased genomic and proteomic screenings using ovarian cancer cell lines of different genetic backgrounds and sensitivities to platinum led to the identification of two key genes upregulated by mifepristone and involved in the unfolded protein response (UPR): the master chaperone of the endoplasmic reticulum (ER), glucose regulated protein (GRP) of 78 kDa, and the CCAAT/enhancer binding protein homologous transcription factor (CHOP). GRP78 and CHOP were upregulated by mifepristone in ovarian cancer cells regardless of p53 status and platinum sensitivity. Further studies revealed that the three UPR-associated pathways, PERK, IRE1α, and ATF6, were activated by mifepristone. Also, the synthetic steroid acutely increased mRNA translation rate, which, if prevented, abrogated the splicing of XBP1 mRNA, a non-translatable readout of IRE1α activation. Moreover, mifepristone increased LC3-II levels due to increased autophagic flux. When the autophagic-lysosomal pathway was inhibited with chloroquine, mifepristone was lethal to the cells. Lastly, doses of proteasome inhibitors that are inadequate to block the activity of the proteasomes, caused cell death when combined with mifepristone; this phenotype was accompanied by accumulation of poly-ubiquitinated proteins denoting proteasome inhibition. The stimulation by mifepristone of ER stress and autophagic flux offers a therapeutic opportunity for utilizing this compound to sensitize ovarian cancer cells to proteasome or lysosome inhibitors. PMID:27233943

  7. Oxathiazolones Selectively Inhibit the Human Immunoproteasome over the Constitutive Proteasome

    PubMed Central

    2014-01-01

    Selective inhibitors for the human immunoproteasome LMP7 (β5i) subunit over the constitutive proteasome hold promise for the treatment of autoimmune and inflammatory diseases and hematologic malignancies. Here we report that oxathiazolones inhibit the immunoproteasome β5i with up to 4700-fold selectivity over the constitutive proteasome, are cell permeable, and inhibit proteasomes inside cells. PMID:24900849

  8. In vivo pharmacodynamic imaging of proteasome inhibition.

    PubMed

    Kimbrel, Erin A; Davis, Tina N; Bradner, James E; Kung, Andrew L

    2009-01-01

    Inhibiting the proteolytic activity of the 26S proteasome has been shown to have selective apoptotic effects on cancer cells and to be clinically efficacious in certain malignancies. There is an unmet medical need for additional proteasome inhibitors, and their development will be facilitated by surrogate markers of proteasome function. Toward this end, ectopic fusion of the destruction domain from ornithine decarboxylase (ODC) to reporter proteins is often used for assessing proteasome function. For luciferase-based reporters, we hypothesized that the oxygen-dependent destruction domain (ODD) from hypoxia-inducible factor 1 alpha (HIF-1 alpha) may provide improved sensitivity over luciferase-ODC, owing to its extremely rapid turnover by the proteasome (HIF-1 alpha has a half-life of less than 5 minutes). In the current study, we show that ODD-luciferase affords a greater dynamic range and faster kinetics than luciferase-ODC in sensing proteasome inhibition in vitro. Importantly, ODD-luciferase also serves as an effective in vivo marker of proteasome function in xenograft tumor models, with inhibition being detected by noninvasive imaging within 3 hours of bortezomib administration. These data establish ODD-luciferase as a surrogate marker of proteasome function that can be used both in vitro and in vivo for the development of novel proteasome inhibitors. PMID:19723471

  9. Effect of addition of FSH, LH and proteasome inhibitor MG132 to in vitro maturation medium on the developmental competence of yak (Bos grunniens) oocytes

    PubMed Central

    2014-01-01

    Background The competence for embryonic development after IVF is low in the yak, therefore, we investigated the effects of supplementation of FSH, LH and the proteasome inhibitor MG132 in IVM media on yak oocyte competence for development after IVF. Methods In Experiment 1, yak cumulus-oocyte complexes (COCs) were in vitro matured (IVM) in TCM-199 with 20% fetal calf serum (FCS), 1 microg/mL estradiol-17beta, and different combinations of LH (50 or 100 IU/mL) and FSH (0, 1, 5, 10 microg/mL) at 38.6 degrees C, 5% CO2 in air for 24 h. Matured oocytes were exposed to frozen–thawed, heparin-capacitated yak sperm. Presumptive zygotes were cultured in SOF medium containing 6 mg/ml BSA, 0.5 mg/mL myoinositol, 3% (v/v) essential amino acids, 1% nonessential amino acids and 100 μg/mL L-glutamine (48 h, 38.5 degrees C, 5% CO2, 5% O2, and 90% N2). In Experiment 2, cumulus cells were collected at the end of IVM to determine FSHR and LHR mRNA expression by real-time PCR. In Experiment 3 and 4, COCs were cultured in the presence or absence of the proteasomal inhibitor MG132 from either 0–6 h or 18–24 h after initiation of maturation. Results The optimum concentration of FSH and LH in IVM media was 5 microg/mL FSH and 50 IU/mL LH which resulted in the greatest cleavage (79.1%) and blastocyst rates (16.1%). Both FSHR and LHR mRNA were detected in yak cumulus cells after IVM. Treatment with MG132 early in maturation reduced (P < 0.05) cleavage and blastocyst rates. Conversely, treatment with MG132 late in maturation improved (P < 0.05) blastocyst rate. Optimal results with MG132 were achieved at a concentration of 10 microM. Conclusions An optimum concentration of FSH and LH in IVM medium, and treatment with MG132 late in maturation can improve yak oocytes competence for development after IVF. PMID:24754924

  10. MLN2238, a proteasome inhibitor, induces caspase-dependent cell death, cell cycle arrest, and potentiates the cytotoxic activity of chemotherapy agents in rituximab-chemotherapy-sensitive or rituximab-chemotherapy-resistant B-cell lymphoma preclinical models.

    PubMed

    Gu, Juan J; Hernandez-Ilizaliturri, Francisco J; Mavis, Cory; Czuczman, Natalie M; Deeb, George; Gibbs, John; Skitzki, Joseph J; Patil, Ritesh; Czuczman, Myron S

    2013-11-01

    To further develop therapeutic strategies targeting the proteasome system, we studied the antitumor activity and mechanisms of action of MLN2238, a reversible proteasome inhibitor, in preclinical lymphoma models. Experiments were conducted in rituximab-chemotherapy-sensitive cell lines, rituximab-chemotherapy-resistant cell lines (RRCL), and primary B-cell lymphoma cells. Cells were exposed to MLN2238 or caspase-dependent inhibitors, and differences in cell viability, alterations in apoptotic protein levels, effects on cell cycle, and the possibility of synergy when combined with chemotherapeutic agents were evaluated. MLN2238 showed more potent dose-dependent and time-dependent cytotoxicity and inhibition of cell proliferation in lymphoma cells than bortezomib. Our data suggest that MLN2238 can induce caspase-independent cell death in RRCL. MLN2238 (and to a much lesser degree bortezomib) reduced RRCL S phase and induced cell cycle arrest in the G2/M phase. Exposure of rituximab-chemotherapy-sensitive cell lines and RRCL to MLN2238 potentiated the cytotoxic effects of gemcitabine, doxorubicin, and paclitaxel and overcame resistance to chemotherapy in RRCL. MLN2238 is a potent proteasome inhibitor active in rituximab-chemotherapy-sensitive and rituximab-chemotherapy-resistant cell models and potentiates the antitumor activity of chemotherapy agents and has the potential of becoming an effective therapeutic agent in the treatment of therapy-resistant B-cell lymphoma. PMID:23995855

  11. The Proteasome Inhibitor Bortezomib Enhances ATRA-Induced Differentiation of Neuroblastoma Cells via the JNK Mitogen-Activated Protein Kinase Pathway

    PubMed Central

    Luo, Peihua; Lin, Meili; Li, Lin; Yang, Bo; He, Qiaojun

    2011-01-01

    Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. Differentiated human NBs are associated with better outcome and lower stage; induction of differentiation is considered to be therapeutically advantageous. All-trans retinoic acid (ATRA) has been shown to induce the differentiation of neuroblastoma (NB) cell lines. The proteasome inhibitor bortezomib inhibits cell growth and angiogenesis in NBs. Here, we investigated the synergistic effect between bortezomib and ATRA in inducing NB cell differentiation in different NB cell lines. Bortezomib combined with ATRA had a significantly enhanced antiproliferative effect. This inhibition was characterized by a synergistic increase in neuronal differentiation. At the same time, the combination therapy showed little neuronal toxicity which was assessed in primary cultures of rat cerebellar granule cells by the MTT assay, PI staining. The combination of bortezomib and ATRA triggered increased differentiation through the activation of proteins, including RARα, RARβ, RARγ, p-JNK and p21, compared with ATRA treatment alone. Using JNK inhibitor SP600125 to block JNK-dependent activity, the combination therapy-induced neuronal differentiation was partially attenuated. In addition, p21 shRNA had no effect on the combination therapy-induced neuronal differentiation. The in vivo antitumor activities were examined in human NB cell xenografts and GFP-labeled human NB cell xenografts. Treatment of human NB cell CHP126-bearing nude mice with ATRA plus bortezomib resulted in more significant tumor growth inhibition than mice treated with either drug alone. These findings provide the rationale for the development of a new therapeutic strategy for NB based on the pharmacological combination of ATRA and bortezomib. PMID:22087283

  12. KRAS Genotype Correlates with Proteasome Inhibitor Ixazomib Activity in Preclinical In Vivo Models of Colon and Non-Small Cell Lung Cancer: Potential Role of Tumor Metabolism.

    PubMed

    Chattopadhyay, Nibedita; Berger, Allison J; Koenig, Erik; Bannerman, Bret; Garnsey, James; Bernard, Hugues; Hales, Paul; Maldonado Lopez, Angel; Yang, Yu; Donelan, Jill; Jordan, Kristen; Tirrell, Stephen; Stringer, Bradley; Xia, Cindy; Hather, Greg; Galvin, Katherine; Manfredi, Mark; Rhodes, Nelson; Amidon, Ben

    2015-01-01

    In non-clinical studies, the proteasome inhibitor ixazomib inhibits cell growth in a broad panel of solid tumor cell lines in vitro. In contrast, antitumor activity in xenograft tumors is model-dependent, with some solid tumors showing no response to ixazomib. In this study we examined factors responsible for ixazomib sensitivity or resistance using mouse xenograft models. A survey of 14 non-small cell lung cancer (NSCLC) and 6 colon xenografts showed a striking relationship between ixazomib activity and KRAS genotype; tumors with wild-type (WT) KRAS were more sensitive to ixazomib than tumors harboring KRAS activating mutations. To confirm the association between KRAS genotype and ixazomib sensitivity, we used SW48 isogenic colon cancer cell lines. Either KRAS-G13D or KRAS-G12V mutations were introduced into KRAS-WT SW48 cells to generate cells that stably express activated KRAS. SW48 KRAS WT tumors, but neither SW48-KRAS-G13D tumors nor SW48-KRAS-G12V tumors, were sensitive to ixazomib in vivo. Since activated KRAS is known to be associated with metabolic reprogramming, we compared metabolite profiling of SW48-WT and SW48-KRAS-G13D tumors treated with or without ixazomib. Prior to treatment there were significant metabolic differences between SW48 WT and SW48-KRAS-G13D tumors, reflecting higher oxidative stress and glucose utilization in the KRAS-G13D tumors. Ixazomib treatment resulted in significant metabolic regulation, and some of these changes were specific to KRAS WT tumors. Depletion of free amino acid pools and activation of GCN2-eIF2α-pathways were observed both in tumor types. However, changes in lipid beta oxidation were observed in only the KRAS WT tumors. The non-clinical data presented here show a correlation between KRAS genotype and ixazomib sensitivity in NSCLC and colon xenografts and provide new evidence of regulation of key metabolic pathways by proteasome inhibition. PMID:26709701

  13. KRAS Genotype Correlates with Proteasome Inhibitor Ixazomib Activity in Preclinical In Vivo Models of Colon and Non-Small Cell Lung Cancer: Potential Role of Tumor Metabolism

    PubMed Central

    Chattopadhyay, Nibedita; Berger, Allison J.; Koenig, Erik; Bannerman, Bret; Garnsey, James; Bernard, Hugues; Hales, Paul; Maldonado Lopez, Angel; Yang, Yu; Donelan, Jill; Jordan, Kristen; Tirrell, Stephen; Stringer, Bradley; Xia, Cindy; Hather, Greg; Galvin, Katherine; Manfredi, Mark; Rhodes, Nelson; Amidon, Ben

    2015-01-01

    In non-clinical studies, the proteasome inhibitor ixazomib inhibits cell growth in a broad panel of solid tumor cell lines in vitro. In contrast, antitumor activity in xenograft tumors is model-dependent, with some solid tumors showing no response to ixazomib. In this study we examined factors responsible for ixazomib sensitivity or resistance using mouse xenograft models. A survey of 14 non-small cell lung cancer (NSCLC) and 6 colon xenografts showed a striking relationship between ixazomib activity and KRAS genotype; tumors with wild-type (WT) KRAS were more sensitive to ixazomib than tumors harboring KRAS activating mutations. To confirm the association between KRAS genotype and ixazomib sensitivity, we used SW48 isogenic colon cancer cell lines. Either KRAS-G13D or KRAS-G12V mutations were introduced into KRAS-WT SW48 cells to generate cells that stably express activated KRAS. SW48 KRAS WT tumors, but neither SW48-KRAS-G13D tumors nor SW48-KRAS-G12V tumors, were sensitive to ixazomib in vivo. Since activated KRAS is known to be associated with metabolic reprogramming, we compared metabolite profiling of SW48-WT and SW48-KRAS-G13D tumors treated with or without ixazomib. Prior to treatment there were significant metabolic differences between SW48 WT and SW48-KRAS-G13D tumors, reflecting higher oxidative stress and glucose utilization in the KRAS-G13D tumors. Ixazomib treatment resulted in significant metabolic regulation, and some of these changes were specific to KRAS WT tumors. Depletion of free amino acid pools and activation of GCN2-eIF2α-pathways were observed both in tumor types. However, changes in lipid beta oxidation were observed in only the KRAS WT tumors. The non-clinical data presented here show a correlation between KRAS genotype and ixazomib sensitivity in NSCLC and colon xenografts and provide new evidence of regulation of key metabolic pathways by proteasome inhibition. PMID:26709701

  14. The cyclin-dependent kinase inhibitor butyrolactone is a potent inhibitor of p21 (WAF1/CIP1 expression).

    PubMed

    Sax, Joanna K; Dash, Bipin C; Hong, Rui; Dicker, David T; El-Deiry, Wafik S

    2002-01-01

    Butyrolactone I (BL) is a competitive inhibitor of ATP for binding and activation of cyclin-dependent kinases and is a potent inhibitor of cell cycle progression. Treatment of H460 human lung and SW480 human colon cancer cells with doses of BL that exceed the Ki for CDK inhibition but which are much lower than doses required to inhibit MAPK, PKA, PKC, or EGFR lead to a rapid significant reduction of endogenous p21 protein expression. BL-dependent inhibition of p21 expression appears to be p53-independent. BL-dependent p21 degradation was blocked by lactacystin, consistent with the hypothesis that there is accelerated p21 proteasomal degradation in the presence of BL. BL also inhibited the p53-dependent increase of p21 protein expression in cells exposed to the DNA damag-ing agent etoposide, and favored a greater G2/M arrest as compared to the non-BL exposed cells. BL accelerated the degradation of exogenously expressed p21 that was not observed with a C-terminal truncated form of p21. Degradation of exogenous p21 led to a shift to G2 accumulation in the cells exposed to BL. We conclude that BL has effects on the cell cycle beyond its role as a CDK inhibitor and can be used as a novel tool to study the mechanism of p21 degradation and the consequences towards p21- dependent checkpoints. PMID:12429914

  15. A mechanistic insight into a proteasome-independent constitutive inhibitor kappaBalpha (IkappaBalpha) degradation and nuclear factor kappaB (NF-kappaB) activation pathway in WEHI-231 B-cells.

    PubMed Central

    Shumway, Stuart D; Miyamoto, Shigeki

    2004-01-01

    Inducible activation of the transcription factor NF-kappaB (nuclear factor kappaB) is classically mediated by proteasomal degradation of its associated inhibitors, IkappaBalpha (inhibitory kappaBalpha) and IkappaBbeta. However, certain B-lymphocytes maintain constitutively nuclear NF-kappaB activity (a p50-c-Rel heterodimer) which is resistant to inhibition by proteasome inhibitors. This activity in the WEHI-231 B-cell line is associated with continual and preferential degradation of IkappaBalpha, which is also unaffected by proteasome inhibitors. Pharmacological studies indicated that there was a correlation between inhibition of IkappaBalpha degradation and constitutive p50-c-Rel activity. Domain analysis of IkappaBalpha by deletion mutagenesis demonstrated that an N-terminal 36-amino-acid sequence of IkappaBalpha represented an instability determinant for constitutive degradation. Moreover, domain grafting studies indicated that this sequence was sufficient to cause IkappaBbeta, but not chloramphenicol acetyltransferase, to be rapidly degraded in WEHI-231 B-cells. However, this sequence was insufficient to target IkappaBbeta to the non-proteasome degradation pathway, suggesting that there was an additional cis-element(s) in IkappaBalpha that was required for complete targeting. Nevertheless, the NF-kappaB pool associated with IkappaBbeta now became constitutively active by virtue of IkappaBbeta instability in these cells. These findings further support the notion that IkappaB instability governs the maintenance of constitutive p50-c-Rel activity in certain B-cells via a unique degradation pathway. PMID:14763901

  16. Could inhibition of the proteasome cause mad cow disease?

    PubMed

    Hooper, Nigel M

    2003-04-01

    The proteasome is the cellular machinery responsible for the degradation of normal and misfolded proteins. Inhibitors of the proteasome are being evaluated as therapeutic agents and recent work suggests that such inhibition might promote the neurotoxic properties of the prion protein (the causative agent of mad cow disease) and its conformational conversion to the infectious form, thus raising the question as to whether proteasome inhibitors might facilitate the development of prion diseases. PMID:12679058

  17. A beta-lactone related to lactacystin induces neurite outgrowth in a neuroblastoma cell line and inhibits cell cycle progression in an osteosarcoma cell line.

    PubMed Central

    Fenteany, G; Standaert, R F; Reichard, G A; Corey, E J; Schreiber, S L

    1994-01-01

    Lactacystin, a microbial natural product, induces neurite outgrowth in Neuro 2A mouse neuroblastoma cells and inhibits progression of synchronized Neuro 2A cells and MG-63 human osteosarcoma cells beyond the G1 phase of the cell cycle. A related beta-lactone, clasto-lactacystin beta-lactone, formally the product of elimination of N-acetylcysteine from lactacystin, is also active, whereas the corresponding clastolactacystin dihydroxy acid is completely inactive. Structural analogs of lactacystin altered only in the N-acetylcysteine moiety are active, while structural or stereochemical modifications of the gamma-lactam ring or the hydroxyisobutyl group lead to partial or complete loss of activity. The inactive compounds do not antagonize the effects of lactacystin in either neurite outgrowth or cell cycle progression assays. The response to lactacystin involves induction of a predominantly bipolar morphology that is maximal 16-32 h after treatment and is distinct from the response to several other treatments that result in morphological differentiation. Neurite outgrowth in response to lactacystin appears to be dependent upon microtubule assembly, actin polymerization, and de novo protein synthesis. The observed structure-activity relationships suggest that lactacystin and its related beta-lactone may act via acylation of one or more relevant target molecule(s) in the cell. Images PMID:8159752

  18. Dinaciclib, a Cyclin-Dependent Kinase Inhibitor Promotes Proteasomal Degradation of Mcl-1 and Enhances ABT-737-Mediated Cell Death in Malignant Human Glioma Cell Lines.

    PubMed

    Jane, Esther P; Premkumar, Daniel R; Cavaleri, Jonathon M; Sutera, Philip A; Rajasekar, Thatchana; Pollack, Ian F

    2016-02-01

    The prognosis for malignant glioma, the most common brain tumor, is still poor, underscoring the need to develop novel treatment strategies. Because glioma cells commonly exhibit genomic alterations involving genes that regulate cell-cycle control, there is a strong rationale for examining the potential efficacy of strategies to counteract this process. In this study, we examined the antiproliferative effects of the cyclin-dependent kinase inhibitor dinaciclib in malignant human glioma cell lines, with intact, deleted, or mutated p53 or phosphatase and tensin homolog on chromosome 10; intact or deleted or p14ARF or wild-type or amplified epidermal growth factor receptor. Dinaciclib inhibited cell proliferation and induced cell-cycle arrest at the G2/M checkpoint, independent of p53 mutational status. In a standard 72-hour 3-[4,5-dimethylthiazol- 2yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H, tetrazolium (MTS) assay, at clinically relevant concentrations, dose-dependent antiproliferative effects were observed, but cell death was not induced. Moreover, the combination of conventional chemotherapeutic agents and various growth-signaling inhibitors with dinaciclib did not yield synergistic cytotoxicity. In contrast, combination of the Bcl-2/Bcl-xL inhibitors ABT-263 (4-[4-[[2-(4-chlorophenyl)-5,5-dimethylcyclohexen-1-yl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-morpholin-4-yl-1-phenylsulfanylbutan-2-yl]amino]-3-(trifluoromethylsulfonyl)phenyl]sulfonylbenzamide) or ABT-737 (4-[4-[[2-(4-chlorophenyl)phenyl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-(dimethylamino)-1-phenylsulfanylbutan-2-yl]amino]-3-nitrophenyl]sulfonylbenzamide) with dinaciclib potentiated the apoptotic response induced by each single drug. The synergistic killing by ABT-737 with dinaciclib led to cell death accompanied by the hallmarks of apoptosis, including an early loss of the mitochondrial transmembrane potential; the release of cytochrome c, smac/DIABLO, and apoptosis-inducing factor

  19. Exposure-safety-efficacy analysis of single-agent ixazomib, an oral proteasome inhibitor, in relapsed/refractory multiple myeloma: dose selection for a phase 3 maintenance study.

    PubMed

    Gupta, Neeraj; Labotka, Richard; Liu, Guohui; Hui, Ai-Min; Venkatakrishnan, Karthik

    2016-06-01

    Background Ixazomib is the first oral, small molecule proteasome inhibitor to reach phase 3 trials. The current analysis characterized the exposure-safety and exposure-efficacy relationships of ixazomib in patients with relapsed/refractory multiple myeloma (MM) with a purpose of recommending an approach to ixazomib dosing for maintenance therapy. Methods Logistic regression was used to investigate relationships between ixazomib plasma exposure (area under the curve/day; derived from individual apparent clearance values from a published population pharmacokinetic analysis) and safety/efficacy outcomes (hematologic [grade ≥ 3 vs ≤ 2] or non-hematologic [grade ≥ 2 vs ≤ 1] adverse events [AEs], and clinical benefit [≥stable disease vs progressive disease]) using phase 1 data in relapsed/refractory MM (NCT00963820; N = 44). Results Significant relationships to ixazomib exposure were observed for five AEs (neutropenia, thrombocytopenia, rash, fatigue, and diarrhea) and clinical benefit (p < 0.05). Dose-response relationships indicated a favorable benefit/risk ratio at 3 mg and 4 mg weekly, which are below the maximum tolerated dose of 5.5 mg. At 3 mg, the model predicted that: 37 % of patients will achieve clinical benefit; incidence of grade ≥ 3 neutropenia and thrombocytopenia will be 10 % and 23 %, respectively; and incidence of grade ≥ 2 rash, fatigue, and diarrhea will be 8 %, 19 %, and 19 %, respectively. Conclusions Based on the findings, patients in the phase 3 maintenance trial will initiate ixazomib at a once-weekly dose of 3 mg, increasing to 4 mg if acceptable tolerability after 4 cycles, to provide maximum clinical benefit balanced with adequate tolerability. PMID:27039387

  20. Obatoclax interacts synergistically with the irreversible proteasome inhibitor carfilzomib in GC- and ABC-DLBCL cells in vitro and in vivo.

    PubMed

    Dasmahapatra, Girija; Lembersky, Dmitry; Son, Minkyeong P; Patel, Hiral; Peterson, Derick; Attkisson, Elisa; Fisher, Richard I; Friedberg, Jonathan W; Dent, Paul; Grant, Steven

    2012-05-01

    Interactions between the irreversible proteasome inhibitor carfilzomib and the pan-BH3 mimetic obatoclax were examined in germinal center (GC)- and activated B-cell-diffuse large B-cell lymphoma (ABC-DLBCL) cells. Cotreatment with minimally toxic concentrations of carfilzomib (i.e., 2-6 nmol/L) and subtoxic concentrations of obatoclax (0.05-2.0 μmol/L) synergistically increased apoptosis in multiple DLBCL cell lines and increased lethality toward primary human DLBCL but not normal CD34(+) cells. Synergistic interactions were associated with sharp increases in caspase-3 activation, PARP cleavage, p-JNK induction, upregulation of Noxa, and AKT dephosphorylation. Combined treatment also diminished carfilzomib-mediated Mcl-1 upregulation whereas immunoprecipitation analysis revealed reduced associations between Bak and Mcl-1/Bcl-xL and Bim and Mcl-1. The carfilzomib/obatoclax regimen triggered translocation, conformational change, and dimerization of Bax and activation of Bak. Genetic interruption of c-jun-NH(2)-kinase (JNK) and Noxa by short hairpin RNA knockdown, ectopic Mcl-1 expression, or enforced activation of AKT significantly attenuated carfilzomib/obatoclax-mediated apoptosis. Notably, coadministration of carfilzomib/obatoclax sharply increased apoptosis in multiple bortezomib-resistant DLBCL models. Finally, in vivo administration of carfilzomib and obatoclax to mice inoculated with SUDHL4 cells substantially suppressed tumor growth, activated JNK, inactivated AKT, and increased survival compared with the effects of single-agent treatment. Together, these findings argue that a strategy combining carfilzomib and obatoclax warrants attention in DLBCL. PMID:22411899

  1. Proteasomal degradation of sphingosine kinase 1 and inhibition of dihydroceramide desaturase by the sphingosine kinase inhibitors, SKi or ABC294640, induces growth arrest in androgen-independent LNCaP-AI prostate cancer cells

    PubMed Central

    McNaughton, Melissa; Pitman, Melissa; Pitson, Stuart M.; Pyne, Nigel J.; Pyne, Susan

    2016-01-01

    Sphingosine kinases (two isoforms termed SK1 and SK2) catalyse the formation of the bioactive lipid sphingosine 1-phosphate. We demonstrate here that the SK2 inhibitor, ABC294640 (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide) or the SK1/SK2 inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole)) induce the proteasomal degradation of SK1a (Mr = 42 kDa) and inhibit DNA synthesis in androgen-independent LNCaP-AI prostate cancer cells. These effects are recapitulated by the dihydroceramide desaturase (Des1) inhibitor, fenretinide. Moreover, SKi or ABC294640 reduce Des1 activity in Jurkat cells and ABC294640 induces the proteasomal degradation of Des1 (Mr = 38 kDa) in LNCaP-AI prostate cancer cells. Furthermore, SKi or ABC294640 or fenretinide increase the expression of the senescence markers, p53 and p21 in LNCaP-AI prostate cancer cells. The siRNA knockdown of SK1 or SK2 failed to increase p53 and p21 expression, but the former did reduce DNA synthesis in LNCaP-AI prostate cancer cells. Moreover, N-acetylcysteine (reactive oxygen species scavenger) blocked the SK inhibitor-induced increase in p21 and p53 expression but had no effect on the proteasomal degradation of SK1a. In addition, siRNA knockdown of Des1 increased p53 expression while a combination of Des1/SK1 siRNA increased the expression of p21. Therefore, Des1 and SK1 participate in regulating LNCaP-AI prostate cancer cell growth and this involves p53/p21-dependent and -independent pathways. Therefore, we propose targeting androgen-independent prostate cancer cells with compounds that affect Des1/SK1 to modulate both de novo and sphingolipid rheostat pathways in order to induce growth arrest. PMID:26934645

  2. Proteasomal degradation of sphingosine kinase 1 and inhibition of dihydroceramide desaturase by the sphingosine kinase inhibitors, SKi or ABC294640, induces growth arrest in androgen-independent LNCaP-AI prostate cancer cells.

    PubMed

    McNaughton, Melissa; Pitman, Melissa; Pitson, Stuart M; Pyne, Nigel J; Pyne, Susan

    2016-03-29

    Sphingosine kinases (two isoforms termed SK1 and SK2) catalyse the formation of the bioactive lipid sphingosine 1-phosphate. We demonstrate here that the SK2 inhibitor, ABC294640 (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide) or the SK1/SK2 inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole)) induce the proteasomal degradation of SK1a (Mr = 42 kDa) and inhibit DNA synthesis in androgen-independent LNCaP-AI prostate cancer cells. These effects are recapitulated by the dihydroceramide desaturase (Des1) inhibitor, fenretinide. Moreover, SKi or ABC294640 reduce Des1 activity in Jurkat cells and ABC294640 induces the proteasomal degradation of Des1 (Mr = 38 kDa) in LNCaP-AI prostate cancer cells. Furthermore, SKi or ABC294640 or fenretinide increase the expression of the senescence markers, p53 and p21 in LNCaP-AI prostate cancer cells. The siRNA knockdown of SK1 or SK2 failed to increase p53 and p21 expression, but the former did reduce DNA synthesis in LNCaP-AI prostate cancer cells. Moreover, N-acetylcysteine (reactive oxygen species scavenger) blocked the SK inhibitor-induced increase in p21 and p53 expression but had no effect on the proteasomal degradation of SK1a. In addition, siRNA knockdown of Des1 increased p53 expression while a combination of Des1/SK1 siRNA increased the expression of p21. Therefore, Des1 and SK1 participate in regulating LNCaP-AI prostate cancer cell growth and this involves p53/p21-dependent and -independent pathways. Therefore, we propose targeting androgen-independent prostate cancer cells with compounds that affect Des1/SK1 to modulate both de novo and sphingolipid rheostat pathways in order to induce growth arrest. PMID:26934645

  3. BcR-induced apoptosis involves differential regulation of C16 and C24-ceramide formation and sphingolipid-dependent activation of the proteasome.

    PubMed

    Kroesen, Bart-Jan; Jacobs, Susan; Pettus, Benjamin J; Sietsma, Hannie; Kok, Jan Willem; Hannun, Yusuf A; de Leij, Lou F M H

    2003-04-25

    In this study, we describe an ordered formation of long- and very long-chain ceramide species in relation to the progression of B-cell receptor (BcR) triggering induced apoptosis. An early and caspase-independent increase in long-chain ceramide species, in which C(16)- ceramide predominated, was observed 6 h after BcR triggering. In contrast, very long-chain ceramide species were generated later, 12-24 h after BcR triggering. The formation of these very long-chain ceramide species, in which C(24)-ceramide predominated, required the activation of effector caspases. BcR-induced formation of long-chain ceramide species resulted in proteasomal activation and degradation of XIAP and subsequent activation of effector caspases, demonstrating an important cell-biological mechanism through which long-chain ceramides may be involved in the progression of BcR triggering induced apoptosis and subsequent formation of very long-chain ceramide species. BcR-induced activation of the proteasome was blocked with ISP-1/myriocin, a potent and selective inhibitor of serine palmitoyl transferase that catalyzes the first and rate-limiting step in the de novo formation of ceramide. Both ISP-1 and clasto-lactacystin beta-lactone, an irreversible inhibitor of the proteasome, prevented BcR cross-linking-induced XIAP degradation. Also, a mutant XIAP lacking the ubiquitin-ligating ring finger motif was completely resistant to proteasome-mediated degradation, and Ramos cells overexpressing XIAP became highly resistant to BcR cross-linking-induced activation of caspases. The formation of C(16)-ceramide in response to BcR cross-linking was found unaltered in XIAP overexpressing Ramos cells, whereas C(24)-ceramide formation was completely abolished. These results demonstrate how de novo generated long-chain ceramide species may be involved in the activation of downstream effector caspases and subsequent formation of very long-chain ceramide species. As such, these results provide novel and

  4. Inhibition of nuclear factor-{kappa}B and target genes during combined therapy with proteasome inhibitor bortezomib and reirradiation in patients with recurrent head-and-neck squamous cell carcinoma

    SciTech Connect

    Van Waes, Carter . E-mail: vanwaesc@nidcd.nih.gov; Chang, Angela A.; Lebowitz, Peter F.; Druzgal, Colleen H.; Chen, Zhong; Elsayed, Yusri A.; Sunwoo, John B.; Rudy, Susan; Morris, John C.; Mitchell, James B.; Camphausen, Kevin; Gius, David; Adams, Julian; Sausville, Edward A.; Conley, Barbara A.

    2005-12-01

    Purpose: To examine the effects the proteasome inhibitor bortezomib (VELCADE) on transcription factor nuclear factor-{kappa}B (NF-{kappa}B) and target genes and the feasibility of combination therapy with reirradiation in patients with recurrent head-and-neck squamous cell carcinoma (HNSCC). Methods and Materials: The tolerability and response to bortezomib 0.6 mg/m{sup 2} and 0.9 mg/m{sup 2} given twice weekly concurrent with daily reirradiation to 50-70 Gy was explored. Blood proteasome inhibition and NF-{kappa}B-modulated cytokines and factors were measured. Proteasome inhibition, nuclear localization of NF-{kappa}B phospho-p65, apoptosis, and expression of NF-{kappa}B-modulated mRNAs were compared in serial biopsies from accessible tumors. Results: The maximally tolerated dose was exceeded, and study was limited to 7 and 2 patients, respectively, given bortezomib 0.6 mg/m{sup 2} and 0.9 mg/m{sup 2}/dose with reirradiation. Grade 3 hypotension and hyponatremia were dose limiting. Mucositis was Grade 3 or less and was delayed. The mean blood proteasome inhibition at 1, 24, and 48 h after 0.6 mg/m{sup 2} was 32%, 16%, and 7% and after 0.9 mg/m{sup 2} was 56%, 26%, and 14%, respectively. Differences in proteasome and NF-{kappa}B activity, apoptosis, and expression of NF-{kappa}B-modulated cell cycle, apoptosis, and angiogenesis factor mRNAs were detected in 2 patients with minor tumor reductions and in serum NF-{kappa}B-modulated cytokines in 1 patient with a major tumor reduction. Conclusions: In combination with reirradiation, the maximally tolerated dose of bortezomib was exceeded at a dose of 0.6 mg/m{sup 2} and the threshold of proteasome inhibition. Although this regimen with reirradiation is not feasible, bortezomib induced detectable differences in NF-{kappa}B localization, apoptosis, and NF-{kappa}B-modulated genes and cytokines in tumor and serum in association with tumor reduction, indicating that other schedules of bortezomib combined with primary

  5. Nanoparticles Exacerbate Both Ubiquitin and Heat Shock Protein Expressions in Spinal Cord Injury: Neuroprotective Effects of the Proteasome Inhibitor Carfilzomib and the Antioxidant Compound H-290/51.

    PubMed

    Sharma, Hari S; Muresanu, Dafin F; Lafuente, Jose V; Sjöquist, Per-Ove; Patnaik, Ranjana; Sharma, Aruna

    2015-10-01

    compounds or proteasome inhibitors are required for neuroprotection in the NP-exposed traumatized group, and (iii) ubiquitin and HSP expressions play a key role in neuronal injury in SCI, not reported earlier. PMID:26126513

  6. Synergism between arsenite and proteasome inhibitor MG132 over cell death in myeloid leukaemic cells U937 and the induction of low levels of intracellular superoxide anion

    SciTech Connect

    Lombardo, Tomás; Cavaliere, Victoria; Costantino, Susana N.; Kornblihtt, Laura; Alvarez, Elida M.; Blanco, Guillermo A.

    2012-02-01

    Increased oxygen species production has often been cited as a mechanism determining synergism on cell death and growth inhibition effects of arsenic-combined drugs. However the net effect of drug combination may not be easily anticipated solely from available knowledge of drug-induced death mechanisms. We evaluated the combined effect of sodium arsenite with the proteasome inhibitor MG132, and the anti-leukaemic agent CAPE, on growth-inhibition and cell death effect in acute myeloid leukaemic cells U937 and Burkitt's lymphoma-derived Raji cells, by the Chou–Talalay method. In addition we explored the association of cytotoxic effect of drugs with changes in intracellular superoxide anion (O{sub 2}{sup −}) levels. Our results showed that combined arsenite + MG132 produced low levels of O{sub 2}{sup −} at 6 h and 24 h after exposure and were synergic on cell death induction in U937 cells over the whole dose range, although the combination was antagonistic on growth inhibition effect. Exposure to a constant non-cytotoxic dose of 80 μM hydrogen peroxide together with arsenite + MG132 changed synergism on cell death to antagonism at all effect levels while increasing O{sub 2}{sup −} levels. Arsenite + hydrogen peroxide also resulted in antagonism with increased O{sub 2}{sup −} levels in U937 cells. In Raji cells, arsenite + MG132 also produced low levels of O{sub 2}{sup −} at 6 h and 24 h but resulted in antagonism on cell death and growth inhibition. By contrast, the combination arsenite + CAPE showed high levels of O{sub 2}{sup −} production at 6 h and 24 h post exposure but resulted in antagonism over cell death and growth inhibition effects in U937 and Raji cells. We conclude that synergism between arsenite and MG132 in U937 cells is negatively associated to O{sub 2}{sup −} levels at early time points after exposure. -- Highlights: ► Arsenic combined cytotoxic and anti-proliferative effects by Chou–Talalay method. ► Cytotoxic effect associated

  7. 26S Proteasome: Hunter and Prey in Auxin Signaling.

    PubMed

    Kong, Xiangpei; Zhang, Liangran; Ding, Zhaojun

    2016-07-01

    Auxin binds to TRANSPORT INHIBITOR RESPONSE 1 and AUXIN SIGNALLING F-BOX proteins (TIR1/AFBs) and promotes the degradation of Aux/IAA transcriptional repressors. The proteasome regulator PROTEASOME REGULATOR1 (PTRE1) has now been shown to be required for auxin-mediated repression of 26S proteasome activity, thus providing new insights into the fine-tuning of the homoeostasis of Aux/IAA proteins and auxin signaling. PMID:27246455

  8. Combined 3D-QSAR, Molecular Docking and Molecular Dynamics Study on Derivatives of Peptide Epoxyketone and Tyropeptin-Boronic Acid as Inhibitors Against the β5 Subunit of Human 20S Proteasome

    PubMed Central

    Liu, Jianling; Zhang, Hong; Xiao, Zhengtao; Wang, Fangfang; Wang, Xia; Wang, Yonghua

    2011-01-01

    An abnormal ubiquitin-proteasome is found in many human diseases, especially in cancer, and has received extensive attention as a promising therapeutic target in recent years. In this work, several in silico models have been built with two classes of proteasome inhibitors (PIs) by using 3D-QSAR, homology modeling, molecular docking and molecular dynamics (MD) simulations. The study resulted in two types of satisfactory 3D-QSAR models, i.e., the CoMFA model (Q2 = 0.462, R2pred = 0.820) for epoxyketone inhibitors (EPK) and the CoMSIA model (Q2 = 0.622, R2pred = 0.821) for tyropeptin-boronic acid derivatives (TBA). From the contour maps, some key structural factors responsible for the activity of these two series of PIs are revealed. For EPK inhibitors, the N-cap part should have higher electropositivity; a large substituent such as a benzene ring is favored at the C6-position. In terms of TBA inhibitors, hydrophobic substituents with a larger size anisole group are preferential at the C8-position; higher electropositive substituents like a naphthalene group at the C3-position can enhance the activity of the drug by providing hydrogen bond interaction with the protein target. Molecular docking disclosed that residues Thr60, Thr80, Gly106 and Ser189 play a pivotal role in maintaining the drug-target interactions, which are consistent with the contour maps. MD simulations further indicated that the binding modes of each conformation derived from docking is stable and in accord with the corresponding structure extracted from MD simulation overall. These results can offer useful theoretical references for designing more potent PIs. PMID:21673924

  9. Sensitization of U937 leukemia cells to doxorubicin by the MG132 proteasome inhibitor induces an increase in apoptosis by suppressing NF-kappa B and mitochondrial membrane potential loss

    PubMed Central

    2014-01-01

    Background The resistance of cancerous cells to chemotherapy remains the main limitation for cancer treatment at present. Doxorubicin (DOX) is a potent antitumor drug that activates the ubiquitin-proteasome system, but unfortunately it also activates the Nuclear factor kappa B (NF-кB) pathway leading to the promotion of tumor cell survival. MG132 is a drug that inhibits I kappa B degradation by the proteasome-avoiding activation of NF-кB. In this work, we studied the sensitizing effect of the MG132 proteasome inhibitor on the antitumor activity of DOX. Methods U937 human leukemia cells were treated with MG132, DOX, or both drugs. We evaluated proliferation, viability, apoptosis, caspase-3, -8, and −9 activity and cleavage, cytochrome c release, mitochondrial membrane potential, the Bcl-2 and Bcl-XL antiapoptotic proteins, senescence, p65 phosphorylation, and pro- and antiapoptotic genes. Results The greatest apoptosis percentage in U937 cells was obtained with a combination of MG132 + DOX. Likewise, employing both drugs, we observed a decrease in tumor cell proliferation and important caspase-3 activation, as well as mitochondrial membrane potential loss. Therefore, MG132 decreases senescence, p65 phosphorylation, and the DOX-induced Bcl-2 antiapoptotic protein. The MG132 + DOX treatment induced upregulation of proapoptotic genes BAX, DIABLO, NOXA, DR4, and FAS. It also induced downregulation of the antiapoptotic genes BCL-XL and SURVIVIN. Conclusion MG132 sensitizes U937 leukemia cells to DOX-induced apoptosis, increasing its anti-leukemic effectiveness. PMID:24495648

  10. YSY01A, a Novel Proteasome Inhibitor, Induces Cell Cycle Arrest on G2 Phase in MCF-7 Cells via ERα and PI3K/Akt Pathways

    PubMed Central

    Xue, Bingjie; Huang, Wei; Yuan, Xia; Xu, Bo; Lou, Yaxin; Zhou, Quan; Ran, Fuxiang; Ge, Zemei; Li, Runtao; Cui, Jingrong

    2015-01-01

    Given that the proteasome is essential for multiple cellular processes by degrading diverse regulatory proteins, inhibition of the proteasome has emerged as an attractive target for anti-cancer therapy. YSY01A is a novel small molecule compound targeting the proteasome. The compound was found to suppress viability of MCF-7 cells and cause limited cell membrane damage as determined by sulforhodamine B assay (SRB) and CytoTox 96® non-radioactive cytotoxicity assay. High-content screening (HCS) further shows that YSY01A treatment induces cell cycle arrest on G2 phase within 24 hrs. Label-free quantitative proteomics (LFQP), which allows extensive comparison of cellular responses following YSY01A treatment, suggests that various regulatory proteins including cell cycle associated proteins and PI3K/Akt pathway may be affected. Furthermore, YSY01A increases p-CDC-2, p-FOXO3a, p53, p21Cip1 and p27Kip1 but decreases p-Akt, p-ERα as confirmed by Western blotting. Therefore, YSY01A represents a potential therapeutic for breast cancer MCF-7 by inducing G2 phase arrest via ERα and PI3K/Akt pathways. PMID:25767601