Activation of the ubiquitin-proteasome system contributes to oculopharyngeal muscular dystrophy through muscle atrophy.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder characterized by progressive weakness and degeneration of specific muscles. OPMD is due to extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). Aggregation of the mutant protein in muscle nuclei is a hallmark of the disease. Previous transcriptomic analyses revealed the consistent deregulation of the ubiquitin-proteasome system (UPS) in OPMD animal models and patients, suggesting a role of this deregulation in OPMD pathogenesis. Subsequent studies proposed that UPS contribution to OPMD involved PABPN1 aggregation. Here, we use a Drosophila model of OPMD to address the functional importance of UPS deregulation in OPMD. Through genome-wide and targeted genetic screens we identify a large number of UPS components that are involved in OPMD. Half dosage of UPS genes reduces OPMD muscle defects suggesting a pathological increase of UPS activity in the disease. Quantification of proteasome activity confirms stronger activity in OPMD muscles, associated with degradation of myofibrillar proteins. Importantly, improvement of muscle structure and function in the presence of UPS mutants does not correlate with the levels of PABPN1 aggregation, but is linked to decreased degradation of muscle proteins. Oral treatment with the proteasome inhibitor MG132 is beneficial to the OPMD Drosophila model, improving muscle function although PABPN1 aggregation is enhanced. This functional study reveals the importance of increased UPS activity that underlies muscle atrophy in OPMD. It also provides a proof-of-concept that inhibitors of proteasome activity might be an attractive pharmacological approach for OPMD.

Flavokawain B targets protein neddylation for enhancing the anti-prostate cancer effect of Bortezomib via Skp2 degradation.

BACKGROUND: Flavokawain B (FKB) has been identified from kava root extracts as a potent apoptosis inducer for inhibiting the growth of various cancer cell lines, including prostate cancer. However, the molecular targets of FKB in prostate cancer cells remain unknown. METHODS: An in vitro NEDD8 Initiation Conjugation Assay was used to evaluate the neddylation inhibitory activity of FKB. Molecular docking and a cellular thermal shift assay were performed to assess the direct interaction between FKB and the NEDD8 activating enzyme (NAE) complex. Protein neddylation, ubiqutination, stability and expression in cells were assessed with immunoprecipitation and Western blotting methods using specific antibodies. Deletion and site specific mutants and siRNAs were used to evaluate deep mechanisms by which FKB induces Skp2 degradation. Cell growth inhibition and apoptosis induction were measured by MTT, ELISA and Western blotting methods. RESULTS: FKB inhibits NEDD8 conjugations to both Cullin1 and Ubc12 in prostate cancer cell lines and Ubc12 neddylation in an in vitro assay. Molecular docking study and a cellular thermal shift assay reveal that FKB interacts with the regulatory subunit (i.e. APP-BP1) of the NAE. In addition, FKB causes Skp2 degradation in an ubiquitin and proteasome dependent manner. Overexpression of dominant-negative cullin1 (1-452), K720R mutant (the neddylation site) Cullin1 or the F-box deleted Skp2 that losses its binding to the Skp1/Cullin1 complex causes the resistance to FKB-induced Skp2 degradation, whereas siRNA knock-down of Cdh1, a known E3 ligase of Skp2 for targeted degradation, didn't attenuate the effect of FKB on Skp2 degradation. These results suggest that degradation of Skp2 by FKB is involved in a functional Cullin1. Furthermore, proteasome inhibitors Bortezomib and MG132 transcriptionally down-regulate the expression of Skp2, and their combinations with FKB result in enhanced inhibitory effects on the growth of prostate cancer cell lines via synergistic down-regulation of Skp2 and up-regulation of p27/Kip1 and p21/WAF1 protein expression. FKB also selectively inhibits the growth of RB deficient cells with high expression of Skp2. CONCLUSION: These findings provide a rationale for further investigating combination of FKB and Bortezomib for treatment of RB deficient, castration-resistant prostate cancer.

The Effects of Proteasome Inhibitors on Telomerase Activity and Regulation in Multiple Myeloma Cells.

The importance of telomerase, the enzyme that maintains telomere length, has been reported in many malignancies in general and in multiple myeloma (MM) in particular. Proteasome inhibitors are clinically used to combat effectively MM. Since the mechanism of action of proteasome inhibitors has not been fully described we sought to clarify its potential effect on telomerase activity (TA) in MM cells. Previously we showed that the first generation proteasome inhibitor bortezomib (Brt) inhibits TA in MM cells by both transcriptional and post-translational mechanisms and has a potential clinical significance. In the current study we focused around the anti- telomerase activity of the new generation of proteasome inhibitors, epoxomicin (EP) and MG-132 in order to clarify whether telomerase inhibition represents a class effect. We have exposed MM cell lines, ARP-1, CAG, RPMI 8226 and U266 to EP or MG and the following parameters were assessed: viability; TA, hTERT expression, the binding of hTERT (human telomerase reverse transcriptase) transcription factors and post-translational modifications. Epoxomicin and MG-132 differentially downregulated the proliferation and TA in all MM cell lines. The downregulation of TA and the expression of hTERT were faster in CAG than in ARP-1 cells. Epoxomicin was more potent than MG-132 and therefore further mechanistic studies were performed using this compound. The inhibition of TA was mainly transcriptionally regulated. The binding of three positive regulator transcription factors: SP1, c-Myc and NF-κB to the hTERT promoter was decreased by EP in CAG cells as well as their total cellular expression. In ARP-1 cells the SP1 and c-MYC binding and protein levels were similarly affected by EP while NF-κB was not affected. Interestingly, the transcription factor WT-1 (Wilms' tumor-1) exhibited an increased binding to the hTERT promoter while its total cellular amount remained unchanged. Our results combined with our previous study of bortezomib define telomerase as a general target for proteasome inhibitors. The inhibitory effect of TA is exerted by several regulatory levels, transcriptional and post translational. SP1, C-Myc and NF-κB were involved in mediating these effects. A novel finding of this study is the role of WT-1 in the regulation of telomerase which appears as a negative regulator of hTERT expression. The results of this study may contribute to future development of telomerase inhibition as a therapeutic modality in MM.

The Role of Proteasome Inhibitor MG132 in 2,4-Dinitrofluorobenzene-Induced Atopic Dermatitis in NC/Nga Mice.

BACKGROUND: Although immunosuppressants for therapy of atopic dermatitis (AD) are still being sought, proteasome inhibitors are also potential candidates for the treatment of AD. Proteasome inhibitors exert various effects by blocking proteasomal degradation and help regulate processes such as apoptosis induction via caspase-9, cell cycle progression via cyclins, NF-κB inactivation via IκB, and downregulation of antigen cross-presentation. The cells targeted by proteasome inhibitors are therefore activated cells undergoing proliferation or differentiation, and antigen-presenting cells carrying out protein degradation. OBJECTIVES: This study investigated the therapeutic effects and side effects of a proteasome inhibitor, MG132, on the treatment of AD. METHODS: AD-like disease in NC/Nga mice housed under specific pathogen-free conditions was induced by repeated application of 2,4-dinitrofluorobenzene (DNFB). Disease progression was evaluated by inflammation score, histopathology, and serum IgE level, and the effects of systemic MG132 administration were investigated. The percentages and absolute numbers for each population of Th1, Th2, and Th17 cells in the axillary lymph nodes were analyzed by flow cytometry. RESULTS: DNFB application increased the expression of a unique major histocompatibility complex class I mutant molecule D/Ldm7 in dendritic cells (DCs), and increased Th1 and Th17 cells in NC/Nga mice. In vivo MG132 administration to NC/Nga mice with DNFB-induced dermatitis reduced Th17 cells but maintained the level of Th1 cells, resulting in the alleviation of dermatitis lesions by decreasing both serum IgE hyperproduction and mast cell migration. To understand the mechanisms maintaining Th1 cell levels following in vivo MG132-administration, we focused on the role of proteasomes regulating D/Ldm7 expression. Interestingly, 20S proteasome activity was higher in NC/Nga DCs than in BALB/c DCs. In vitro MG132 administration partially increased D/Ldm7 expression in a dose-dependent manner during DC maturation, and induced IFN-γ production from autoreactive CD8+ T cells but not from CD4+ T cells following coculturing with D/Ldm7-upregulated DCs. CONCLUSION: Although MG132 administration temporarily alleviated AD pathogenesis in NC/Nga mice, prolonged MG132 treatment may result in immunopathogenesis leading to chronic AD due to its side effect of maintaining Th1 levels via autoreactive CD8+ T cells.

Proteasome Inhibitor Carbobenzoxy-L-Leucyl-L-Leucyl-L-Leucinal (MG132) Enhances Therapeutic Effect of Paclitaxel on Breast Cancer by Inhibiting Nuclear Factor (NF)-κB Signaling.

BACKGROUND Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132), a peptide aldehyde proteasome inhibitor, can inhibit tumor progression by inactivating nuclear factor (NF)-κB signaling. Paclitaxel (PTX) is part of a routine regimen for the treatment of breast cancer. However, activation of the NF-κB pathway after treatment with PTX confers insensitivity to this drug. This study investigated the potential effect of MG132 as a co-treatment with PTX against breast cancer, and clarifies the underlying molecular mechanisms. MATERIAL AND METHODS Breast cancer cells were treated with PTX, MG132, or PTX plus MG132, and the therapeutic effects were evaluated phenotypically. A mouse model of breast cancer was used to determine the combined effect of PTX plus MG132 in vivo. RESULTS Treatment with PTX plus MG132 suppressed aggressive phenotypes of breast cancer cells more effectively than PTX alone. Consistently, MG132 also enhanced the suppressive effect of PTX on tumor growth in C57BL/6 mice. Significantly, activation of the NF-κB pathway by PTX was attenuated by MG132. CONCLUSIONS Based on our findings, we suggest the application of MG132 in clinical practice in combination with PTX for the treatment of breast cancer.

Proteasome inhibitor MG132 enhances the antigrowth and antimetastasis effects of radiation in human nonsmall cell lung cancer cells.

The current treatment for advanced nonsmall cell lung cancer (NSCLC) remains unsatisfactory due to resistance to chemotherapy and ionizing radiation. The ubiquitin-proteasome system (UPS) regulates multiple cellular processes that are crucial for the proliferation and survival of all kinds of cells. Carbobenzoxyl-leucinyl-leucinyl-leucinal-H (MG132), a specific and selective reversible inhibitor of the 26S proteasome, represents a novel approach for cancer therapy. However, whether MG132 can potentiate the effect of radiation against the growth and metastasis of NSCLC is not clear. We found that MG132 inhibited the proliferation of human NSCLC cell lines (A549 and H1299) in a dose- and time-dependent manner by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Then MG132 at a nontoxic dose (100 nM) was selected for following studies. Pretreatment of A549 and H1299 cells with 100 nM MG132 before ionizing radiation (IR) potentiated the anticancer effect of IR. Moreover, pretreatment with 100 nM MG132 before IR-enhanced radiation induced cell cycle arrest by decreased CyclinD1 but increased Wee1 expression in A549 and H1299 cells. In addition, pretreatment of MG132 combined with IR significantly suppressed cell migration and invasion abilities in NSCLC cell lines, which was accompanied by decreased expression of matrix metalloproteinase (MMP)-2 and MMP-9 in NSCLC cell lines. Taken together, our results demonstrate that MG132 enhances the antigrowth and antimetastatic effects of irradiation in NSCLC cells by modulating expression of cell cycle and invasion- related genes.

Novel drug targets and molecular mechanisms for sarcopenia based on systems biology.

Sarcopenia is a major public health concern among older adults, leading to disabilities, falls, fractures, and mortality. This study aimed to elucidate the pathophysiological mechanisms of sarcopenia and identify potential therapeutic targets using systems biology approaches. RNA-seq data from muscle biopsies of 24 sarcopenic and 29 healthy individuals from a previous cohort were analysed. Differential expression, gene set enrichment, gene co-expression network, and topology analyses were conducted to identify target genes implicated in sarcopenia pathogenesis, resulting in the selection of 6 hub genes (PDHX, AGL, SEMA6C, CASQ1, MYORG, and CCDC69). A drug repurposing approach was then employed to identify new pharmacological treatment options for sarcopenia (clofibric-acid, troglitazone, withaferin-a, palbociclib, MG-132, bortezomib). Finally, validation experiments in muscle cell line (C2C12) revealed MG-132 and troglitazone as promising candidates for sarcopenia treatment. Our approach, based on systems biology and drug repositioning, provides insight into the molecular mechanisms of sarcopenia and offers potential new treatment options using existing drugs.

Potential role for Nrf2 activation in the therapeutic effect of MG132 on diabetic nephropathy in OVE26 diabetic mice.

Oxidative stress is a major cause of diabetic nephropathy. Upregulation of the key antioxidative transcription factor, nuclear factor-erythroid 2-related factor 2 (Nrf2), was found to prevent the development of diabetic nephropathy. The present study was designed to explore the therapeutic effect of Nrf2 induced by proteasomal inhibitor MG132 at a low dose (10 µg/kg) on diabetic nephropathy. Transgenic type 1 diabetic (OVE26) mice displayed renal dysfunction with albuminuria by 3 mo of age, at which time MG132 treatment was started. After 3-mo treatment with MG132, renal function, morphology, and biochemical changes were examined with real-time PCR, Western blotting, and immunohistochemical examination. Compared with age-matched, nontreated diabetic mice, MG132-treated diabetic mice showed significant improvements in terms of renal structural and functional alterations. These therapeutic effects were associated with increased Nrf2 expression and transcriptional upregulation of Nrf2-regulated antioxidants. Mechanistic study using human renal tubular HK11 cells confirmed the role of Nrf2, as silencing the Nrf2 gene with its specific siRNA abolished MG132 prevention of high-glucose-induced profibrotic response. Furthermore, diabetes was found to significantly increase proteasomal activity in the kidney, an effect that was significantly attenuated by 3 mo of treatment with MG132. These results suggest that MG132 upregulates Nrf2 function via inhibition of diabetes-increased proteasomal activity, which can provide the basis for the therapeutic effect of MG132 on the kidney against diabetes-induced oxidative damage, inflammation, fibrosis, and eventual dysfunction.

ALLN rescues an in vitro excitatory synaptic transmission deficit in Lis1 mutant mice.

LIS1 gene mutations lead to a rare neurological disorder, classical lissencephaly, characterized by brain malformations, mental retardation, seizures, and premature death. Mice heterozygous for Lis1 (Lis1(+/-)) exhibit cortical malformations, defects in neuronal migration, increased glutamate-mediated synaptic transmission, and spontaneous electrographic seizures. Recent work demonstrated that in utero treatment of Lis1(+/-) mutant dams with ALLN, a calpain inhibitor, partially rescues neuronal migration defects in the offspring. Given the challenges of in utero drug administration, we examined the therapeutic potential of ALLN on postnatal lissencephalic cells. Voltage- and current-clamp studies were performed with acute hippocampal slices obtained from Lis1 mutant mice and age-matched littermate control mice. Specifically, we determined whether postnatal ALLN treatment can reverse excitatory synaptic transmission deficits, namely, an increase in spontaneous and miniature excitatory postsynaptic current (EPSC) frequency, on CA1 pyramidal neurons observed in tissue slices from Lis1(+/-) mice. We found that acute application of ALLN restored spontaneous and miniature EPSC frequencies to wild-type levels without affecting inhibitory postsynaptic synaptic current. Furthermore, Western blot analysis of protein expression, including proteins involved in excitatory synaptic transmission, demonstrated that ALLN blocks the cleavage of the calpain substrate αII-spectrin but does not rescue Lis1 protein levels in Lis1(+/-) mutants.

Autophagy is increased in laminin α2 chain-deficient muscle and its inhibition improves muscle morphology in a mouse model of MDC1A.

Congenital muscular dystrophy caused by laminin α2 chain deficiency (also known as MDC1A) is a severe and incapacitating disease, characterized by massive muscle wasting. The ubiquitin-proteasome system plays a major role in muscle wasting and we recently demonstrated that increased proteasomal activity is a feature of MDC1A. The autophagy-lysosome pathway is the other major system involved in degradation of proteins and organelles within the muscle cell. However, it remains to be determined if the autophagy-lysosome pathway is dysregulated in muscular dystrophies, including MDC1A. Using the dy(3K)/dy(3K) mouse model of laminin α2 chain deficiency and MDC1A patient muscle, we show here that expression of autophagy-related genes is upregulated in laminin α2 chain-deficient muscle. Moreover, we found that autophagy inhibition significantly improves the dystrophic dy(3K)/dy(3K) phenotype. In particular, we show that systemic injection of 3-methyladenine (3-MA) reduces muscle fibrosis, atrophy, apoptosis and increases muscle regeneration and muscle mass. Importantly, lifespan and locomotive behavior were also greatly improved. These findings indicate that enhanced autophagic activity is pathogenic and that autophagy inhibition holds a promising therapeutic potential in the treatment of MDC1A.

Therapeutic effect of MG-132 on diabetic cardiomyopathy is associated with its suppression of proteasomal activities: roles of Nrf2 and NF-κB.

MG-132, a proteasome inhibitor, can upregulate nuclear factor (NF) erythroid 2-related factor 2 (Nrf2)-mediated antioxidative function and downregulate NF-κB-mediated inflammation. The present study investigated whether through the above two mechanisms MG-132 could provide a therapeutic effect on diabetic cardiomyopathy in the OVE26 type 1 diabetic mouse model. OVE26 mice develop hyperglycemia at 2-3 wk after birth and exhibit albuminuria and cardiac dysfunction at 3 mo of age. Therefore, 3-mo-old OVE26 diabetic and age-matched control mice were intraperitoneally treated with MG-132 at 10 µg/kg daily for 3 mo. Before and after MG-132 treatment, cardiac function was measured by echocardiography, and cardiac tissues were then subjected to pathological and biochemical examination. Diabetic mice showed significant cardiac dysfunction, including increased left ventricular systolic diameter and wall thickness and decreased left ventricular ejection fraction with an increase of the heart weight-to-tibia length ratio. Diabetic hearts exhibited structural derangement and remodeling (fibrosis and hypertrophy). In diabetic mice, there was also increased systemic and cardiac oxidative damage and inflammation. All of these pathogenic changes were reversed by MG-132 treatment. MG-132 treatment significantly increased the cardiac expression of Nrf2 and its downstream antioxidant genes with a significant increase of total antioxidant capacity and also significantly decreased the expression of IκB and the nuclear accumulation and DNA-binding activity of NF-κB in the heart. These results suggest that MG-132 has a therapeutic effect on diabetic cardiomyopathy in OVE26 diabetic mice, possibly through the upregulation of Nrf2-dependent antioxidative function and downregulation of NF-κB-mediated inflammation.

Leupeptin maintains redox homeostasis via targeting ROS-autophagy-inflammatory axis in LPS-stimulated macrophages and cytokines dichotomy in Con-A challenged lymphocyte.

Information regarding cellular anti-inflammatory and immunomodulatory attributes of leupeptin with respect to modulation of perturbed macrophage function and lymphocytes has not yet been delineated, particularly in the context of ROS-cytokines-autophagy-inflammatory signalling cascades. Therefore, the present study identified the attributes and mechanisms of leupeptin, from actinomycetes, in relation to excessive oxidative stress mediated disrupted immune homeostasis and inflammatory mechanism in activated macrophages and lymphocytes. Results revealed that leupeptin treatment showed noticeable inhibition in the production of NO, ROS, mitochondrial membrane potential and phagocytosis activity in LPS-stimulated macrophages. These findings were accompanied by reduction in TNF-α, IL-1ß, IL-6, IFN-γ/IL-10 ratio, endopeptidases, oxidative effectors (Cox-2, IL-5, IL-15, IL-17, COX-2), iNOS with concomitant increase in Arg 1, Msr 1 and Mrc - 1exprssion in leupeptin treatment. Additionally, compared to LPS-challenged cells, marked alleviation in MDC, lysotracker staining, beclin-1, LC3B expression, and enhanced p62 levels in leupeptin exposed cells indicate the reversal of impaired autophagy flux. Subsequently, oxi-inflammatory signalling analysis demonstrated p-PTEN, p-NF-κB, p-PI3K, p-Akt, p-p38, and ERK1/2 upregulation decisively thwarted by leupeptin administration. In silico analysis further implied its target selectivity to these cascades. Furthermore, decreased proliferation index and Th1, Th2/IL-10 cytokines ratio in mitogen-challenged splenic lymphocytes confers its role in mitigating unwarranted inflammation mediated by disrupted regulation of adaptive immune cells. Together, these findings signify the attributes of leupeptin as an alternative anti-inflammatory strategy and affirm it as a promising natural entity to modulate immune-mediated response during inflammatory disorder.

MG132 alleviates liver injury induced by intestinal ischemia/reperfusion in rats: involvement of the AhR and NFκB pathways.

BACKGROUND: MG132 is a potent antioxidant and has been reported to play a protective role in ischemia/reperfusion (I/R) of many organs. Recent studies have shown that the Aryl hydrocarbon receptor (AhR) may play a beneficial role in I/R of many organs and an AhR agonist has been implicated in an anti-inflammatory role. MG132 might function as an AhR agonist through proteasome inhibition, possibly through the inhibition of NFκB. Herein, we hypothesized that MG132 may play a protective role in liver injury induced by intestinal I/R and we analyzed the expression behavior of AhR and NFκB to determine whether the two factors play a role in intestinal I/R. MATERIALS AND METHODS: Thirty-two Sprague-Dawley rats were divided into four groups: control, I/R, MG132 control, and MG132 pretreatment. The I/R and MG132 pretreatment groups were subjected to mesenteric arterial ischemia for 1 h and reperfusion for 3 h. The control and MG132 control groups underwent surgical preparation including isolation of the superior mesenteric artery (SMA) without occlusion. The MG132 control and MG132 pretreatment groups were subjected to intraperitoneal administration of 0.5 mg/kg MG132 30 min before surgery. We collected serum specimens to measure TNF-α, IL-6, liver tissue levels of malondialdehyde (MDA), AhR, and cyp1a2; NFκB, IκBα, and ICAM-1 were also tested. Histologic changes of liver and intestine were subsequently evaluated. RESULTS: Compared with the control group, significant increases in MDA, NFκB, and ICAM-1 levels were accompanied by decreases in AhR, cyp1a2, and IκBα expression in the liver in the I/R group, which is consistent with liver and intestinal tissue injury. MG132 blocked the alterations of the indicators above. There were no changes in the MG132 control group compared with the control group in the indicators above. CONCLUSIONS: This study demonstrated that MG132 has a significant effect in protection against liver injury induced by intestinal I/R, which may be due to modulation of the AhR and NFκB pathways.

MG132 Induces Progerin Clearance and Improves Disease Phenotypes in HGPS-like Patients' Cells.

Progeroid syndromes (PS), including Hutchinson-Gilford Progeria Syndrome (HGPS), are premature and accelerated aging diseases, characterized by clinical features mimicking physiological aging. Most classical HGPS patients carry a de novo point mutation within exon 11 of the LMNA gene encoding A-type lamins. This mutation activates a cryptic splice site, leading to the production of a truncated prelamin A, called prelamin A ∆50 or progerin, that accumulates in HGPS cell nuclei and is a hallmark of the disease. Some patients with PS carry other LMNA mutations and are named "HGPS-like" patients. They produce progerin and/or other truncated prelamin A isoforms (∆35 and ∆90). We previously found that MG132, a proteasome inhibitor, induced progerin clearance in classical HGPS through autophagy activation and splicing regulation. Here, we show that MG132 induces aberrant prelamin A clearance and improves cellular phenotypes in HGPS-like patients' cells other than those previously described in classical HGPS. These results provide preclinical proof of principle for the use of a promising class of molecules toward a potential therapy for children with HGPS-like or classical HGPS.

Treatment with MG132 prevents spontaneous activation of rat oocyte in culture and promotes embryonic development after intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) is an effective reproductive technique for obtaining rat offspring using preserved sperm with low or no motility. However, rat oocytes undergo spontaneous activation immediately after retrieval from the oviduct and poorly develop after ICSI unless it is performed quickly. Here, we evaluated whether treatment with MG132, the proteasome inhibitor, suppresses the spontaneous activation of oocytes before and during ICSI. After retrieval from the oviducts, the rate of development into morula and blastocyst from the oocytes cultured in vitro for 1 h prior to ICSI significantly decreased compared with that from the control oocytes subject to ICSI without culture (7% versus 36%). However, a higher proportion of oocytes treated with MG132 for 0, 1, and 3 h before and during ICSI developed into morulae and blastocysts (70%, 60%, and 52%, respectively). Offspring were obtained from oocytes treated with MG132 for 0 and 1 h before and during ICSI (percentage: 31%). Altogether, MG132 could suppress the spontaneous activation of rat oocytes and increase embryonic development after ICSI.

p53-Independent up-regulation of a TRAIL receptor DR5 by proteasome inhibitors: a mechanism for proteasome inhibitor-enhanced TRAIL-induced apoptosis.

Gliomas are the most common brain tumors in adults and account for more than half of all brain tumors. Despite intensive clinical investigations, average survival for the patients harboring the malignancy has not been significantly improved. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), shown to have potent and cancer-selective killing activity, has drawn considerable attention as a promising anti-cancer therapy. In an attempt to develop TRAIL as an anti-cancer therapy for gliomas, tumor suppressor activity of TRAIL was assessed using human glioma cell lines such as U373MG, U343MG, U87MG and LN18. U343MG, U87MG and LN18 cells were susceptible to TRAIL; however, U373MG cells were completely refractory to TRAIL. Resistance to the applied therapies is a key issue in cancer treatment; thus, various combination treatments were evaluated using U373MG cells to identify a better regimen. Unlike Doxorubicin, Etoposide, Actinomycin D and Wortmannin, a proteasome inhibitor MG132 significantly enhanced TRAIL-induced apoptosis. Similarly, other proteasome inhibitors, including Lactacystin, Proteasome inhibitor I and Velcade (Bortezomib), also enhanced apoptotic activity of TRAIL. Among these proteasome inhibitors, Velcade, the only approved drug, was as effective as MG132 in enhancing TRAIL-induced apoptosis. Both Velcade and MG132 increased the protein levels of DR5, a TRAIL receptor known to be up-regulated by p53, in U373MG cells where p53 is mutated. Our data indicate that proteasome inhibitors up-regulate DR5 in a p53-independent manner and a combination therapy comprising TRAIL and Velcade become a better treatment regimen for gliomas.

The mammalian target of rapamycin inhibitor RAD001 (everolimus) synergizes with chemotherapeutic agents, ionizing radiation and proteasome inhibitors in pre-B acute lymphocytic leukemia.

BACKGROUND: Despite incremental improvements in outcomes for patients with acute lymphoblastic leukemia, significant numbers of patients still die from this disease. Mammalian target of rapamycin inhibitors have shown potential in vitro and in vivo as therapeutic agents against a range of tumors including acute lymphoblastic leukemia. DESIGN AND METHODS: Flow cytometry was used to evaluate drug-induced cell death in acute lymphoblastic leukemia cell lines and patients' samples. Human xenografts in immunocompromised mice were used to assess the in vivo effects of selected combinations. Pharmacological inhibitors and lentiviral small interfering ribonucleic acid knock-down of p53 were used to investigate the mechanism of cell killing involved. RESULTS: Synergistic interactions between RAD001 and cytotoxic agents were demonstrated in vitro and in vivo, with increased caspase-dependent killing. RAD001 suppressed p53 and p21 responses, while suppression of p53 did not prevent killing, indicating p53 independence. RAD001 and cytotoxic agents activated the JUN N-terminal kinase pathway and the combination further increased JUN N-terminal kinase activation. JUN N-terminal kinase inhibition reduced synergistic cell killing by cytotoxic agents and RAD001 in pre-B acute lymphoblastic leukemia cell lines and patients' samples. Bortezomib and MG132, which activate the JUN N-terminal kinase pathway, also synergized with RAD001 in killing pre-B acute lymphoblastic leukemia cells. Killing was greater when RAD001 was combined with proteasome inhibitors than with cytotoxic drugs. CONCLUSIONS: These observations suggest that combining mammalian target of rapamycin inhibitors with conventional chemotherapy or selected novel agents has the potential to improve clinical responses in patients with pre-B acute lymphoblastic leukemia.

Prevention of muscle disuse atrophy by MG132 proteasome inhibitor.

INTRODUCTION: Our goal was to determine whether in vivo administration of the proteasome inhibitor MG132 can prevent muscle atrophy caused by hindlimb unloading (HU). METHODS: Twenty-seven NMRI mice were assigned to a weight-bearing control, a 6-day HU, or a HU+MG132 (1 mg/kg/48 h) treatment group. RESULTS: Gastrocnemius wasting was significantly less in HU+MG132 mice (-6.7 ± 2.0%) compared with HU animals (-12.6 ± 1.1%, P = 0.011). HU was also associated with an increased expression of MuRF-1 (P = 0.006), MAFbx (P = 0.001), and USP28 (P = 0.027) mRNA, whereas Nedd4, E3α, USP19, and UBP45 mRNA did not change significantly. Increases in MuRF-1, MAFbx, and USP28 mRNA were largely repressed after MG132 administration. ß5 proteasome activity tended to increase in HU (+16.7 ± 6.1%, P = 0.086). Neither ß1 and ß2 proteasome activities nor ubiquitin-conjugated proteins were changed by HU. CONCLUSIONS: Our results indicate that in vivo administration of MG132 partially prevents muscle atrophy associated with disuse and highlight an unexpected regulation of MG132 proteasome inhibitor on ubiquitin-ligases.

Proteasome inhibition alleviates prolonged moderate compression-induced muscle pathology.

BACKGROUND: The molecular mechanism initiating deep pressure ulcer remains to be elucidated. The present study tested the hypothesis that the ubiquitin proteasome system is involved in the signalling mechanism in pressure-induced deep tissue injury. METHODS: Adult Sprague Dawley rats were subjected to an experimental compression model to induce deep tissue injury. The tibialis region of the right hind limb was subjected to 100 mmHg of static pressure for six hours on each of two consecutive days. The compression pressure was continuously monitored by a three-axial force transducer within the compression indentor. The left hind limb served as the intra-animal control. Muscle tissues underneath the compressed region were collected and used for analyses. RESULTS: Our results demonstrated that the activity of 20S proteasome and the protein abundance of ubiquitin and MAFbx/atrogin-1 were elevated in conjunction with pathohistological changes in the compressed muscle, as compared to control muscle. The administration of the proteasome inhibitor MG132 was found to be effective in ameliorating the development of pathological histology in compressed muscle. Furthermore, 20S proteasome activity and protein content of ubiquitin and MAFbx/atrogin-1 showed no apparent increase in the MG132-treated muscle following compression. CONCLUSION: Our data suggest that the ubiquitin proteasome system may play a role in the pathogenesis of pressure-induced deep tissue injury.

Intracochlear perfusion of leupeptin and z-VAD-FMK: influence of antiapoptotic agents on gunshot-induced hearing loss.

The therapeutic efficiency of cochlear infusion of two anti-apoptotic substances: a potent calpain inhibitor, leupeptin and a caspase inhibitor, z-VAD-FMK was evaluated in guinea pigs after a gunshot noise-induced trauma (170 dB SPL). A preliminary study showed that hair cell apoptosis appeared within 7 days of the noise trauma. For each animal, one of the cochleae was perfused directly starting 1 h after the trauma with leupeptin or z-VAD-FMK for 7 days via a mini-osmotic pump whereas the other cochlea was untreated. ABR threshold shifts were measured over a 14-day recovery period. The functional hearing study was supplemented by histological analysis. Two days after the trauma significant differences were observed between threshold shifts in the z-VAD-FMK-treated and the non-treated ears. Cochleograms showed that hair cell losses were significantly lower in z-VAD-FMK-treated ears. Regarding the leupeptin treatment, no significant difference between treated and non-treated ears was observed. This work indicates that early direct infusion of z-VAD-FMK into the cochlea accelerates hearing recovery and reduces hair cell loss after gunshot noise-induced trauma. These results suggest that the gunshot noise-induced trauma may involve the caspase pathway rather than the calpain pathway in the apoptotic process.