Showing papers on "MG132 published in 2017"

Uncovering the SUMOylation and ubiquitylation crosstalk in human cells using sequential peptide immunopurification.

Abstract: Crosstalk between the SUMO and ubiquitin pathways has recently been reported. However, no approach currently exists to determine the interrelationship between these modifications. Here, we report an optimized immunoaffinity method that permits the study of both protein ubiquitylation and SUMOylation from a single sample. This method enables the unprecedented identification of 10,388 SUMO sites in HEK293 cells. The sequential use of SUMO and ubiquitin remnant immunoaffinity purification facilitates the dynamic profiling of SUMOylated and ubiquitylated proteins in HEK293 cells treated with the proteasome inhibitor MG132. Quantitative proteomic analyses reveals crosstalk between substrates that control protein degradation, and highlights co-regulation of SUMOylation and ubiquitylation levels on deubiquitinase enzymes and the SUMOylation of proteasome subunits. The SUMOylation of the proteasome affects its recruitment to promyelocytic leukemia protein (PML) nuclear bodies, and PML lacking the SUMO interacting motif fails to colocalize with SUMOylated proteasome further demonstrating that this motif is required for PML catabolism.

Interaction between Cucumber mosaic virus 2b protein and plant catalase induces a specific necrosis in association with proteasome activity.

Abstract: Cucumber mosaic virus (CMV) can induce a specific necrosis on Arabidopsis through the interaction between the CMV 2b protein and host catalase, in which the ubiquitin–proteasome pathway may be involved. We previously reported that the CMV 2b protein, the viral RNA silencing suppressor, interacted with the H2O2 scavenger catalase (CAT3), leading to necrosis on CMV-inoculated Arabidopsis leaves. We here confirmed that CMV could more abundantly accumulate in the CAT3-knockout mutant (cat3), and that CAT3 makes host plants a little more tolerant to CMV. We also found that the necrosis severity is not simply explained by a high level of H2O2 given by the lack of CAT3, because the recombinant CMV, CMV-N, induced much milder necrosis in cat3 than in the wild type, suggesting some specific mechanism for the necrosis induction. To further characterize the 2b-inducing necrosis in relation to its binding to CAT3, we conducted the agroinfiltration experiments to overexpress CAT3 and 2b in N. benthamiana leaves. The accumulation levels of CAT3 were higher when co-expressed with the CMV-N 2b (N2b) than with CMV-Y 2b (Y2b). We infer that N2b made a more stable complex with CAT3 than Y2b did, and the longevity of the 2b–CAT3 complex seemed to be important to induce necrosis. By immunoprecipitation (IP) with an anti-ubiquitin antibody followed by the detection with anti-CAT3 antibodies, we detected a higher molecular-weight smear and several breakdown products of CAT3 among the IP-proteins. In addition, the proteasome inhibitor MG132 treatment could actually increase the accumulation levels of CAT3. This study suggests that the host proteasome pathway is, at least partially, responsible for the degradation of CAT3, which is manifested in CMV-infected tissues.

Sulforaphane Induced Apoptosis via Promotion of Mitochondrial Fusion and ERK1/2-Mediated 26S Proteasome Degradation of Novel Pro-survival Bim and Upregulation of Bax in Human Non-Small Cell Lung Cancer Cells.

Abstract: Previous studies in our laboratory showed that sulforaphane (SFN) induced apoptosis by sustained activation of extracellular regulated protein kinases 1/2 (ERK1/2). However, the underlying mechanisms associated with SFN-induced apoptosis and downstream cascades which are modulated by ERK1/2 were not elucidated. Herein we demonstrated for the first time that alteration of mitochondrial dynamics contributed to SFN-induced apoptosis in human non-small cell lung cancer (NSCLC) cells. Reports showed that protein Bim not only induced apoptosis but also promoted proliferation under certain circumstances. We found that Bim was related to cell growth in NSCLC cells. Pro-survival Bim downregulation was shown to induce apoptosis in response to SFN. Further, Using the ERK1/2 inhibitor, PD98059, we found that SFN upregulated Bax and downregulated Bim through the ERK1/2-dependent signaling pathway. Furthermore, SFN activated ERK1/2 to increase 26S proteasome activity to degrade Bim, while the proteasome inhibitor MG132 reversed this effect. Therefore, SFN phosphorylated ERK1/2 and activated the proteasome system leading to the degradation of Bim, which contributed to apoptosis in NSCLC cells. These findings provided a novel insight into SFN-related therapeutics in cancer treatment.

AtAIRP2 E3 Ligase Affects ABA and High-Salinity Responses by Stimulating Its ATP1/SDIRIP1 Substrate Turnover

Abstract: AtAIRP2 is a cytosolic RING-type E3 ubiquitin ligase that positively regulates an abscisic acid (ABA) response in Arabidopsis (Arabidopsis thaliana). Yeast two-hybrid screening using AtAIRP2 as bait identified ATP1 (AtAIRP2 Target Protein1) as a substrate of AtAIRP2. ATP1 was found to be identical to SDIRIP1, which was reported recently to be a negative factor in ABA signaling and a target protein of the RING E3 ligase SDIR1. Accordingly, ATP1 was renamed ATP1/SDIRIP1. A specific interaction between AtAIRP2 and ATP1/SDIRIP1 and ubiquitination of ATP1/SDIRIP1 by AtAIRP2 were demonstrated in vitro and in planta. The turnover of ATP1/SDIRIP1 was regulated by AtAIRP2 in cell-free degradation and protoplast cotransfection assays. The ABA-mediated germination assay of 35S:ATP1/SDIRIP1-RNAi/atairp2 double mutant progeny revealed that ATP1/SDIRIP1 acts downstream of AtAIRP2. AtAIRP2 and SDIR1 reciprocally complemented the ABA- and salt-insensitive germination phenotypes of sdir1 and atairp2 mutants, respectively, indicating their combinatory roles in seed germination. Subcellular localization and bimolecular fluorescence complementation experiments in the presence of MG132, a 26S proteasome inhibitor, showed that AtAIRP2 and ATP1/SDIRIP1 were colocalized to the cytosolic spherical body, which lies in close proximity to the nucleus, in tobacco (Nicotiana benthamiana) leaf cells. The 26S proteasome subunits RPN12a and RPT1 and the molecular chaperones HSP70 and HSP101 were colocalized to these discrete punctae-like structures. These results raised the possibility that AtAIRP2 and ATP1/SDIRIP1 interact in the cytosolic spherical compartment. Collectively, our data suggest that the down-regulation of ATP1/SDIRIP1 by AtAIRP2 and SDIR1 RING E3 ubiquitin ligases is critical for ABA and high-salinity responses during germination in Arabidopsis.

Degradation of Mcl-1 through GSK-3β Activation Regulates Apoptosis Induced by Bufalin in Non-Small Cell Lung Cancer H1975 Cells.

Abstract: Background/Aims: Mcl-1, an anti-apoptotic Bcl-2 family member, is often overexpressed in non-small cell lung cancer (NSCLC). Bufalin has been reported to induce apoptosis in various tumor cells. However, there is no report showing that bufalin could downregulate Mcl-1 expression in NSCLC. Methods: Cell proliferation was analyzed by cell counting kit-8 (CCK-8) assay in H1975 cells. Cell apoptosis was detected by flow cytometry. Mcl-1 mRNA was detected by RT-PCR. The expression of apoptosis-associated proteins in H1975 cells was detected by western blotting. The levels of Mcl-1 ubiquitination and NOXA were analyzed by Immunoprecipitation assay. Results: Cell growth was inhibited by bufalin in a time and dose-dependent manner. Bufalin induced apoptosis in NSCLC cells by activating caspase cascades and downregulating Mcl-1 expression. However, overexpression of Mcl-1 diminished bufalin-induced apoptosis. Furthermore, bufalin did not reduce Mcl-1 mRNA expression in H1975 cells, but strongly promoted Mcl-1 protein degradation. Proteasome inhibitor MG132 markedly prevented the degradation of Mcl-1 and blocked bufalin-induced Mcl-1 reduction. Bufalin did not significantly affect NOXA protein levels, but downregulated the expression of p-GSK-3β. GSK-3 inhibitor and GSK-3β siRNA resulted in increased levels of Mcl-1 and reversed the bufalin-induced Mcl-1 degradation. Conclusion: Bufalin induced cell apoptosis in H1975 cells may be through downregulation of Mcl-1. Proteasomal degradation of Mcl-1 via GSK-3β activation was involved in bufalin-induced apoptosis.

Antiglioma effects of N6-isopentenyladenosine, an endogenous isoprenoid end product, through the downregulation of epidermal growth factor receptor.

Abstract: Malignant gliomas are highly dependent on the isoprenoid pathway for the synthesis of lipid moieties critical for cell proliferation. The isoprenoid derivative N6-isopentenyladenosine (iPA) displays pleiotropic biological effects, including a direct anti-tumor activity in several tumor models. The antiglioma effects of iPA was then explored in U87MG cells both in vitro and grafted in mice and the related molecular mechanism confirmed in primary derived patients' glioma cells. iPA powerfully inhibited tumor cell growth and induced caspase-dependent apoptosis through a mechanism involving a marked accumulation of the pro-apoptotic BIM protein and inhibition of EGFR. Indeed, activating AMPK following conversion into its iPAMP active form, iPA stimulated EGFR phosphorylation and ubiquitination along a proteasome-mediated pathway which was responsible for receptor degradation and its downstream signaling pathways inhibition, including the STAT3, ERK and AKT cascade. The inhibition of AMPK by compound C prevented iPA-mediated phosphorylation of EGFR, known to precede receptor loss. As expected the block of EGFR degradation, by exposure to the proteasome inhibitor MG132, significantly reduced iPA-induced cell death. Given the importance of receptor degradation in iPA-mediated cytotoxicity, we also documented that the EGFR expression levels in a panel of primary glioma cells confers them a high sensitivity to iPA treatment. In conclusion our study provides the first evidence of iPA antiglioma effect. Indeed, as glioma is driven by aberrant signaling of growth factor receptors, particularly the EGFR, iPA, alone or in association with EGFR targeted therapies, might be a promising therapeutic tool to achieve a potent anti-tumoral effect.

Proteasome inhibitor-induced cleavage of HSP90 is mediated by ROS generation and caspase 10-activation in human leukemic cells

Abstract: Heat shock protein 90 (HSP90) is a molecular chaperone that supports the stability of client proteins. The proteasome is one of the targets for cancer therapy, and studies are underway to use proteasome inhibitors as anti-cancer drugs. In this study, we found that HSP90 was cleaved to a 55kDa protein after treatment with proteasome inhibitors including MG132 in leukemia cells but was not cleaved in other tissue-derived cells. HSP90 has two major isoforms (HSP90α and HSP90β), and both were cleaved by MG132 treatment. MG132 treatment also induced a decrease in HSP90 client proteins. MG132 treatment generated ROS, and the cleavage of HSP90 was blocked by a ROS scavenger, N-acetylcysteine (NAC). MG132 activated several caspases, and the activation was reduced by pretreatment with NAC. Based on an inhibitor study, the cleavage of HSP90 induced by MG132 was dependent on caspase 10 activation. Furthermore, active recombinant caspase 10 induced HSP90 cleavage in vitro. MG132 upregulated VDUP-1 expression and reduced the GSH levels implying that the regulation of redox-related proteins is involved. Taken all together, our results suggest that the cleavage of HSP90 by MG132 treatment is mediated by ROS generation and caspase 10 activation. HSP90 cleavage may provide an additional mechanism involved in the anti-cancer effects of proteasome inhibitors.

Nucleolar Proteome Analysis and Proteasomal Activity Assays Reveal a Link between Nucleolus and 26S Proteasome in A. thaliana.

Abstract: In all eukaryotic cells, the nucleolus is functionally and structurally linked to rRNA synthesis and ribosome biogenesis. This compartment contains as well factors involved in other cellular activities, but the functional interconnection between non-ribosomal activities and the nucleolus (structure and function) still remains an open question. Here, we report a novel mass spectrometry analysis of isolated nucleoli from Arabidopsis thaliana plants using the FANoS (Fluorescence Assisted Nucleolus Sorting) strategy. We identified many ribosome biogenesis factors (RBF) and proteins non-related with ribosome biogenesis, in agreement with the recognized multi-functionality of the nucleolus. Interestingly, we found that 26S proteasome subunits localize in the nucleolus and demonstrated that proteasome activity and nucleolus organization are intimately linked to each other. Proteasome subunits form discrete foci in the disorganized nucleolus of nuc1.2 plants. Nuc1.2 protein extracts display reduced proteasome activity in vitro compared to WT protein extracts. Remarkably, proteasome activity in nuc1.2 is similar to proteasome activity in WT plants treated with proteasome inhibitors (MG132 or ALLN). Finally, we show that MG132 treatment induces disruption of nucleolar structures in WT but not in nuc1.2 plants. Altogether, our data suggest a functional interconnection between nucleolus structure and proteasome activity.

N-acetyl-L-cysteine protects astrocytes against proteotoxicity without recourse to glutathione

Abstract: N-acetyl-l-cysteine (NAC) exhibits protective properties in brain injury models and has undergone a number of clinical trials. Most studies of NAC have focused on neurons. However, neuroprotection may be complemented by the protection of astrocytes because healthier astrocytes can better support the viability of neurons. Here, we show that NAC can protect astrocytes against protein misfolding stress (proteotoxicity), the hallmark of neurodegenerative disorders. Although NAC is thought to be a glutathione precursor, NAC protected primary astrocytes from the toxicity of the proteasome inhibitor MG132 without eliciting any increase in glutathione. Furthermore, glutathione depletion failed to attenuate the protective effects of NAC. MG132 elicited a robust increase in the folding chaperone heat shock protein 70 (Hsp70), and NAC mitigated this effect. Nevertheless, three independent inhibitors of Hsp70 function ablated the protective effects of NAC, suggesting that NAC may help preserve Hsp70 chaperone activity and improve protein quality control without need for Hsp70 induction. Consistent with this view, NAC abolished an increase in ubiquitinated proteins in MG132-treated astrocytes. However, NAC did not affect the loss of proteasome activity in response to MG132, demonstrating that it boosted protein homeostasis and cell viability without directly interfering with the efficacy of this proteasome inhibitor. The thiol-containing molecules l-cysteine and d-cysteine both mimicked the protective effects of NAC, whereas the thiol-lacking molecule N-acetyl-S-methyl-l-cysteine failed to exert protection or blunt the rise in ubiquitinated proteins. Collectively, these findings suggest that the thiol group in NAC is required for its effects on glial viability and protein quality control.

Melatonin reduces endoplasmic reticulum stress and corneal dystrophy-associated TGFBIp through activation of endoplasmic reticulum-associated protein degradation.

Abstract: Endoplasmic reticulum (ER) stress is emerging as a factor for the pathogenesis of granular corneal dystrophy type 2 (GCD2). This study was designed to investigate the molecular mechanisms underlying the protective effects of melatonin on ER stress in GCD2. Our results showed that GCD2 corneal fibroblasts were more susceptible to ER stress-induced death than were wild-type cells. Melatonin significantly inhibited GCD2 corneal cell death, caspase-3 activation, and poly (ADP-ribose) polymerase 1 cleavage caused by the ER stress inducer, tunicamycin. Under ER stress, melatonin significantly suppressed the induction of immunoglobulin heavy-chain-binding protein (BiP) and activation of inositol-requiring enzyme 1α (IRE1α), and their downstream target, alternative splicing of X-box binding protein 1(XBP1). Notably, the reduction in BiP and IRE1α by melatonin was suppressed by the ubiquitin-proteasome inhibitor, MG132, but not by the autophagy inhibitor, bafilomycin A1, indicating involvement of the ER-associated protein degradation (ERAD) system. Melatonin treatment reduced the levels of transforming growth factor-β-induced protein (TGFBIp) significantly, and this reduction was suppressed by MG132. We also found reduced mRNA expression of the ERAD system components HRD1 and SEL1L, and a reduced level of SEL1L protein in GCD2 cells. Interestingly, melatonin treatments enhanced SEL1L levels and suppressed the inhibition of SEL1L N-glycosylation caused by tunicamycin. In conclusion, this study provides new insights into the mechanisms by which melatonin confers its protective actions during ER stress. The results also indicate that melatonin might have potential as a therapeutic agent for ER stress-related diseases including GCD2.

Proteasome Inhibitor MG132 Enhances Cisplatin-Induced Apoptosis in Osteosarcoma Cells and Inhibits Tumor Growth

Abstract: Although cisplatin has been shown to be an integral part of chemotherapy regimen in osteosarcoma (OS) treatment, toxicity issues and chemoresistance have hindered therapeutic development for OS. Exploring novel combination therapy methods is needed to circumvent the limitations of cisplatin alone. The proteasome inhibitor MG132 has shown antitumor effects in many solid tumors. However, little is known about its effects in combination with cisplatin in OS cells. In this study, we examined the effects of MG132 in combination with cisplatin in human OS cells (MG-63 and HOS). MG132 and cisplatin were applied to OS cells, respectively or jointly. The results demonstrated that MG132 markedly inhibited cell viability in a dose- and time-dependent manner, whereas viability of osteoblast cells was not affected, suggesting a selective toxicity of MG132 to cancerous cells. Mechanistically, MG132 arrested cells in the G₂/M phase in association with increased p21waf1 and induced cell apoptosis, which was accompanied by cleaved PARP. In addition to its apoptotic effect alone, MG132 significantly enhanced cisplatin-induced apoptosis in OS cells. Furthermore, cell viability of the combined application of 10 μM MG132 and 5 μg/ml cisplatin was markedly inhibited compared to that of the individual application. These events were accompanied by the downregulation of NF-κB, mitochondrial antiapoptotic protein Bcl-xL, and PI3K/Akt, which play a key role in cell survival. Finally, combination treatment of MG132 and cisplatin showed more antiproliferative effect than the single treatment in OS xenograft models. In summary, we concluded that MG132 interacted synergistically with cisplatin, which raised the possibility that combining the two drugs may represent a novel strategy in OS.

Geniposide accelerates proteasome degradation of Txnip to inhibit insulin secretion in pancreatic β-cells

Abstract: To analyze the role of geniposide in the protein degradation of Txnip and to determine the impact of Txnip on geniposide-regulated GSIS in pancreatic INS-1 cells. The content of Txnip protein was measured by western blot; insulin content and glucose uptake were determined by ELISA; and knockdown of Txnip was the method of RNA interference. Glucose induces a rapid increase in Txnip protein, and geniposide accelerates the degradation of Txnip via proteasome pathway in the presence of high glucose (25 mM) in INS-1 pancreatic β-cells. And MG132, a proteasomal inhibitor, potentiates glucose uptake, metabolism (ATP production) and glucose-stimulated insulin secretion (GSIS) in high-glucose (25 mM)-treated INS-1 cells, but geniposide significantly prevents these effects. Furthermore, the combination of geniposide and Txnip knockdown shows substantial synergistic effects to reduce glucose uptake, metabolism and GSIS in high-glucose (25 mM)-treated INS-1 cells. Txnip protein played an essential role in glucose uptake, metabolism and GSIS, and geniposide could accelerate the degradation via proteasome pathway in high-glucose-treated pancreatic INS-1 cells.

Neuroprotective Effects of Protein Tyrosine Phosphatase 1B Inhibition against ER Stress-Induced Toxicity.

Abstract: Several lines of evidence suggest that endoplasmic reticulum (ER) stress plays a critical role in the pathogenesis of many neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis Protein tyrosine phosphatase 1B (PTP1B) is known to regulate the ER stress signaling pathway, but its role in neuronal systems in terms of ER stress remains largely unknown Here, we showed that rotenone-induced toxicity in human neuroblastoma cell lines and mouse primary cortical neurons was ameliorated by PTP1B inhibition Moreover, the increase in the level of ER stress markers (eIF2α phosphorylation and PERK phosphorylation) induced by rotenone treatment was obviously suppressed by concomitant PTP1B inhibition However, the rotenone-induced production of reactive oxygen species (ROS) was not affected by PTP1B inhibition, suggesting that the neuroprotective effect of the PTP1B inhibitor is not associated with ROS production Moreover, we found that MG132-induced toxicity involving proteasome inhibition was also ameliorated by PTP1B inhibition in a human neuroblastoma cell line and mouse primary cortical neurons Consistently, downregulation of the PTP1B homologue gene in Drosophila mitigated rotenone- and MG132-induced toxicity Taken together, these findings indicate that PTP1B inhibition may represent a novel therapeutic approach for ER stress-mediated neurodegenerative diseases

MDM2 mediates fibroblast activation and renal tubulointerstitial fibrosis via a p53-independent pathway.

Abstract: It is well recognized that murine double minute gene 2 (MDM2) plays a critical role in cell proliferation and inflammatory processes during tumorigenesis. It is also reported that MDM2 is expressed in glomeruli and involved in podocyte injury. However, whether MDM2 is implicated in renal fibrosis remains unclear. Here we investigated the role of MDM2 in tubulointerstitial fibrosis (TIF). By immunohistochemical staining and Western blotting we confirmed that MDM2 is upregulated in the tubulointerstitial compartment in patients with TIF and unilateral urethral obstruction (UUO) mice, which mainly originates from myofibroblasts. Consistently, in vitro MDM2 is increased in TGF-β1-treated fibroblasts, one of the major sources of collagen-producing myofibroblasts during TIF, along with fibroblast activation. Importantly, genetic deletion of MDM2 significantly attenuates fibroblast activation. We then analyzed the possible downstream signaling of MDM2 during fibroblast activation. p53-dependent pathway is the classic downstream signaling of MDM2, and Nutlin-3 is a small molecular inhibitor of MDM2-p53 interaction. To our surprise, Nutlin-3 could not ameliorate fibroblast activation in vitro and TIF in UUO mice. However, we found that Notch1 signaling is attenuated during fibroblast activation, which could be markedly rescued by MDM2 knockdown. Overexpression of intracellular domain of Notch1 (NICD) by plasmid could obviously minimize fibroblast activation induced by TGF-β1. In addition, the degradation of NICD is strikingly suppressed by PYR-41, an inhibitor of ubiquitin-activating enzyme E1, and proteasome inhibitor MG132. Taken together, our findings provide the first evidence that MDM2 is involved in fibroblast activation and TIF, which associates with Notch1 ubiquitination and proteasome degradation.

Treatment with proteasome inhibitor bortezomib decreases organic anion transporting polypeptide (OATP) 1B3-mediated transport in a substrate-dependent manner.

Abstract: OATP1B1 and OATP1B3 mediate hepatic uptake of many drugs (e.g., statins) and can mediate transporter-mediated drug-drug-interactions (DDIs). Bortezomib is the first-in-class proteasome inhibitor drug approved by the U. S. Food and Drug Administration for the treatment of multiple myeloma. The potential of bortezomib to cause OATP-mediated DDIs has not been assessed. The current study investigated the involvement of the ubiquitin-proteasome system (UPS) in OATP1B1 and OATP1B3 degradation and determined the effects of proteasome inhibitors on OATP1B1- and OATP1B3-mediated transport. Co-immunoprecipitation of FLAG-OATP1B1/1B3 and HA-ubiquitin was observed in human embryonic kidney (HEK) 293 cells co-transfected with FLAG-tagged OATP1B1/OATP1B3 and hemagglutinin (HA)-tagged ubiquitin, suggesting that OATP1B1 and OATP1B3 can be ubiquitin-modified. Although blocking proteasome activity by bortezomib treatment (50 nM, 7 h) increased the endogenous ubiquitin-conjugated FLAG-OATP1B1 and FLAG-OATP1B3 in HEK293-FLAG-OATP1B1 and-OATP1B3 cells, such treatment did not affect the total protein levels of OATP1B1 and OATP1B3, suggesting that the UPS plays a minor role in degradation of OATP1B1 and OATP1B3 under current constitutive conditions. Pretreatment with bortezomib (50-250 nM, 2-7 h) significantly decreased transport of [3H]CCK-8, a specific OATP1B3 substrate, in HEK293-OATP1B3 and human sandwich-cultured hepatocytes (SCH). However, bortezomib pretreatment had negligible effects on the transport of [3H]E217βG and [3H]pitavastatin, dual substrates of OATP1B1 and OATP1B3, in HEK293-OATP1B1/1B3 cells and/or human SCH. Compared with vehicle control treatment, bortezomib pretreatment significantly decreased the maximal transport velocity (Vmax) of OATP1B3-mediated transport of CCK-8 (92.25 ± 14.2 vs. 133.95 ± 15.5 pmol/mg protein/min) without affecting the affinity constant (Km) values. Treatment with other proteasome inhibitors MG132, epoxomicin, and carfilzomib also significantly decreased OATP1B3-mediated [3H]CCK-8 transport. In summary, the current studies for the first time report ubiquitination of OATP1B1 and OATP1B3 and the apparent substrate-dependent inhibitory effect of bortezomib on OATP1B3-mediated transport. The data suggest that bortezomib has a low risk of causing OATP-mediated DDIs.

Proteasome inhibitor MG132 induces thyroid cancer cell apoptosis by modulating the activity of transcription factor FOXO3a.

Abstract: Proteasome inhibitors are promising antitumor drugs with preferable cytotoxicity in malignant cells and have exhibited clinical efficiency in several hematologic malignancies. P53-dependent apoptosis has been reported to be a major mechanism underlying. However, apoptosis can also be found in cancer cells with mutant-type p53, suggesting the involvement of p53-independent mechanism. Tumor suppressor forkhead Box O3 is another substrate of proteasomal degradation, which also functions partially through inducing apoptosis. The aim of this study was to explore the effect of proteasome inhibition on the expression and activity of forkhead Box O3 in thyroid cancer cells. Using flow cytometry, western blot, immunofluorescence staining and quantitative RT-PCR assays, we assessed proteasome inhibitor MG132-induced apoptosis in thyroid cancer cells and its effect on the expression and activity of forkhead Box O3. The resulted showed that MG132 induced significant apoptosis, and caused the accumulation of p53 protein in both p53 wild-type and mutant-type thyroid cancer cell lines, whereas the proapoptotic targets of p53 were transcriptionally upregulated only in the p53 wild-type cells. Strikingly, upon MG132 administration, the accumulation and nuclear translocation of transcription factor forkhead Box O3 as well as transcriptional upregulation of its proapoptotic target genes were found in thyroid cancer cells regardless of p53 status. Cell apoptosis was enhanced by ectopic overexpression while attenuated by silencing of forkhead Box O3. Altogether, we demonstrated that proteasome inhibitor MG132 induces thyroid cancer cell apoptosis at least partially through modulating forkhead Box O3 activity.

Proteasome-mediated degradation of collagen III by cortisol in amnion fibroblasts.

Abstract: Rupture of fetal membranes (ROM) can initiate parturition at both term and preterm. Collagen III in the compact layer of the amnion contributes to the tensile strength of fetal membranes. However, the upstream signals triggering collagen III degradation remain mostly elusive. In this study, we investigated the role of cortisol regenerated by 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) in collagen III degradation in human amnion fibroblasts with an aim to seek novel targets for the prevention of preterm premature ROM (pPROM)-elicited preterm birth. Human amnion tissue and cultured amnion tissue explants and amnion fibroblasts were used to study the regulation of collagen III, which is composed of three identical 3α 1 chains (COL3A1), by cortisol. Cortisol decreased COL3A1 protein but not mRNA abundance in a concentration-dependent manner. Cortisone also decreased COL3A1 protein, which was blocked by 11β-HSD1 inhibition. The reduction in COL3A1 protein by cortisol was not affected by a transcription inhibitor but was further enhanced by a translation inhibitor. Autophagic pathway inhibitor chloroquine or siRNA-mediated knock-down of ATG7, an essential protein for autophagy, failed to block cortisol-induced reduction in COL3A1 protein abundance, whereas proteasome pathway inhibitors MG132 and bortezomib significantly attenuated cortisol-induced reduction in COL3A1 protein abundance. Moreover, cortisol increased COL3A1 ubiquitination and the reduction of COL3A1 protein by cortisol was blocked by PYR-41, a ubiquitin-activating enzyme inhibitor. Conclusively, cortisol regenerated in amnion fibroblasts may be associated with ROM at parturition by reducing collagen III protein abundance through a ubiquitin-proteasome pathway.

Hyperglycemia triggers HIPK2 protein degradation

Abstract: Homeodomain interacting protein kinase-2 (HIPK2) is an evolutionary conserved kinase that modulates several key molecular pathways to restrain tumor growth and induce p53-depending apoptotic cell-death in response to anticancer therapies. HIPK2 silencing in cancer cells leads to chemoresistance and cancer progression, in part due to p53 inhibition. Recently, hyperglycemia has been shown to reduce p53 phosphorylation at serine 46 (Ser46), the target residue of HIPK2, thus impairing p53 apoptotic function. Here we asked whether hyperglycemia could, upstream of p53, target HIPK2. We focused on the effect of high glucose (HG) on HIPK2 protein stability and the underlying mechanisms. We found that HG reduced HIPK2 protein levels, therefore impairing HIPK2-induced p53 apoptotic activity. HG-triggered HIPK2 protein downregulation was rescued by both proteasome inhibitor MG132 and by protein phosphatase inhibitors Calyculin A (CL-A) and Okadaic Acid (OA). Looking for the phosphatase involved, we found that protein phosphatase 2A (PP2A) induced HIPK2 degradation, as evidenced by directly activating PP2A with FTY720 or by silencing PP2A with siRNA in HG condition. The effect of PP2A on HIPK2 protein degradation could be in part due to hypoxia-inducible factor-1 (HIF-1) activity which has been previously shown to induce HIPK2 proteasomal degradation through several ubiquitin ligases. Validation analysed performed with HIF-1α dominant negative or with silencing of Siah2 ubiquitin ligase clearly showed rescue of HG-induced HIPK2 degradation. These findings demonstrate how hyperglycemia, through a complex protein cascade, induced HIPK2 downregulation and consequently impaired p53 apoptotic activity, revealing a novel link between diabetes/obesity and tumor resistance to therapies.

Ubiquitin-proteasome system modulates zygotic genome activation in early mouse embryos and influences full-term development.

Abstract: Maternal RNA/protein degradation and zygotic genome activation (ZGA), occurring during maternal-to-zygotic transition (MZT), are the first essential events for the development of pre-implantation embryos. Previously, we have shown the importance of the ubiquitin-proteasome system (UPS) for initiation of minor ZGA at the 1-cell stage of mouse embryos. However, little is known about the mechanism of involvement of the UPS-degraded maternal proteins in ZGA. In this study, we investigated the effect of inhibiting maternal protein degradation by the reversible proteasome inhibitor, MG132, on post-implantation development and ZGA regulation during early cleavage stages. Our study revealed that zygotic transcription by RNA polymerase II (Pol II) at the 1-cell stage was delayed and the full-term development was affected by transient proteasome inhibition during 1 to 9 h post-insemination (hpi). Furthermore, we found that the transient inhibition of proteasome activity at the 2-cell stage delayed the onset of transcription of some major ZGA genes. These results support the model hypothesizing the requirement of sequential degradation of maternal proteins by UPS for the proper onset of ZGA and normal progression of MZT in early mouse embryos.

Proteasome inhibitor MG132 modulates inflammatory pain by central mechanisms in adjuvant arthritis.

Abstract: Aims In rheumatoid arthritis (RA), pain and inflammation are initial symptoms followed by various degrees of bone and cartilage destruction. Previously, we have shown that reversible proteasome inhibitor MG132 attenuates pain and joint inflammation in a rat model of adjuvant-arthritis. Our present study aims to study the effects of MG132 on molecular changes in the dorsal root ganglia (DRG) and in the spinal cord (SC) using the same animal model. Methods Arthritis was induced by heat-killed Mycobacterium butyricum in rats. The expression of substance P (SP) was analyzed by quantitative reverse transcription polymerase chain reaction and immunohistochemistry in DRG and in the SC. The nuclear factor-κB (NF-κB) DNA-binding activity in the SC was analyzed by electromobility shift assay. Results Arthritic rats treated daily with MG132 demonstrated a marked reduction of SP gene expression in the DRG and number of SP-positive cells was reduced. In the spinal cord of arthritic rats elevated SP messenger RNA levels were normalized and NF-κB-DNA-binding activity was down-regulated in arthritic rats treated with MG132. Conclusion Our results indicate that proteasome inhibitor MG132 attenuates pain in adjuvant arthritis by targeting the sensory neuropeptide substance P in the peripheral and central nervous systems. These effects may be mediated through the inhibition of NF-κB activation.

Propofol Provides Cardiac Protection by Suppressing the Proteasome Degradation of Caveolin-3 in Ischemic/Reperfused Rat Hearts.

Abstract: The mechanisms underlying propofol's cardioprotective role remain elusive. Caveolin-3 (Cav-3) has been shown to mediate both opioids- and volatile anesthetics-induced cardioprotection against ischemia/reperfusion (I/R) injury. We hypothesize that the cardioprotective role of propofol is mediated through Cav-3 and its regulation of PI3K/Akt/GSK3β signal pathway. Rats or H9c2 cardiomyocytes were exposed to propofol before I/R or simulated ischemia/reperfusion (SI/R). Propofol pretreatment significantly decreased left ventricle infarct size in vivo (P < 0.05) and terminal deoxynucleotidyl transferase nick-end labeling-positive cells both in vivo and in vitro (P < 0.05), along with an increased Cav-3 protein expression and binding of Cav-3 to p85-subunit of PI3K. No significant change in Cav-3 mRNA expression in left ventricle tissues was found in either I/R or propofol-treated groups. Methyl-β-cyclodextrin or Cav-3 siRNA was used to knockdown Cav-3 expression in vitro, which virtually abolished propofol-induced cardiac protection and PI3K/Akt/GSK3β pathway activation. In contrast, MG132, a proteasome inhibitor, could significantly restore SI/R-induced Cav-3 decrease. It is concluded that Cav-3 mediates propofol-induced cardioprotection against I/R injury and the relevant PI3K/Akt/GSK3β activation. The downregulation of Cav-3 under SI/R may be caused by proteasome degradation, and this process can be prevented by propofol.

Regulation of anti-apoptotic Bcl-2 family protein Mcl-1 by S6 kinase 2.

Abstract: The anti-apoptotic Bcl-2 family protein myeloid cell leukemia-1 (Mcl-1) plays an important role in breast cancer cell survival and chemoresistance. We have previously shown that knockdown of the 40S ribosomal protein S6 kinase-2 (S6K2), which acts downstream of the mechanistic target of rapamycin complex 1 (mTORC1), enhanced breast cancer cell death by apoptotic stimuli. The increase in cell death by S6K2 depletion was partly due to inactivation of Akt. In the present study, we investigated if S6K2 regulates Mcl-1, which acts downstream of Akt. Silencing of S6K2 but not S6K1 in T47D cells decreased Mcl-1 level, and potentiated apoptosis induced by TRAIL and doxorubicin. Knockdown of S6K2 also decreased the level of anti-apoptotic Bcl-xl. Depletion of the tumor suppressor protein PDCD4 (programmed cell death 4), which regulates translation of several anti-apoptotic proteins, reversed downregulation of Bcl-xl but not Mcl-1 and failed to reverse the effect of S6K2 knockdown on potentiation of doxorubicin-induced apoptosis. Downregulation of Mcl-1 by S6K2 knockdown was partly restored by the proteasome inhibitor MG132. Overexpression of catalytically-active Akt or knockdown of glycogen synthase kinase-3 (GSK3)-β, a substrate for Akt, had little effect on Mcl-1 downregulation caused by S6K2 deficiency. Silencing of S6K2 increased the level of c-Jun N-terminal kinase (JNK) and knockdown of JNK1 increased basal Mcl-1 level and partly reversed the effect of S6K2 knockdown on Mcl-1 downregulation. JNK1 knockdown also had a modest effect in attenuating the increase in doxorubicin-induced apoptosis caused by S6K2 deficiency. These results suggest that S6K2 regulates apoptosis via multiple mechanisms, and involves both Akt and JNK.

P‐Glycoprotein Inhibition Sensitizes Human Breast Cancer Cells to Proteasome Inhibitors

Abstract: Although effective for the treatment of hematological malignancies, the FDA approved proteasome inhibitors bortezomib and carfilzomib have limited efficacy in solid tumors including triple negative breast cancer (TNBC). Chemotherapy is the only option for treating TNBC due to the absence of specific therapeutic targets. Therefore, development of new TNBC therapeutic strategies has been warranted. We studied whether P-glycoprotein (P-gp) inhibition could sensitize TNBC cells to proteasome inhibitors. When verapamil, a P-gp inhibitor, was combined with the proteasome inhibitor MG132, bortezomib, or carfilzomib, the cytotoxic effects and apoptosis in TNBC MDA-MB-231 cells were enhanced, compared to each treatment alone. Furthermore, addition of verapamil improved proteasome-inhibitory properties of MG132, bortezomib, or carfilzomib in MDA-MB-231 cells, as shown by the increased accumulation of ubiquitinated proteins and proteasome substrates such as IκBα and p27kip1 . Additionally, when nicardipine, another P-gp inhibitor, was combined with bortezomib or carfilzomib, enhanced inhibition of MDA-MB-231 cell proliferation was observed. These findings indicate that P-gp inhibitors could sensitize TNBC cells to structurally and functionally diverse proteasome inhibitors and might provide new treatment strategy for TNBC. J. Cell. Biochem. 118: 1239-1248, 2017. © 2016 Wiley Periodicals, Inc.

Blocking of the Ubiquitin-Proteasome System Prevents Inflammation-Induced Bone Loss by Accelerating M-CSF Receptor c-Fms Degradation in Osteoclast Differentiation.

Abstract: Anti-osteoporotic activity of a blocker of the ubiquitin-proteasome system, bortezomib, has known to be achieved by directly opposed action in increased bone formation by osteoblasts and in decreased bone destruction by osteoclasts. However, the mechanisms underlying the proteasome blocker inhibition of osteoclast differentiation and function are not fully understood. Here, we observed that proteasome inhibitors, such as MG132 and bortezomib, in osteoclasts accelerated the degradation of c-Fms, a cognate receptor of macrophage colony-stimulating factor (M-CSF), and did not affect the amount of receptor activator of nuclear factor kappa-B (RANK), a receptor of receptor activator of nuclear factor kappa-B ligand (RANKL). c-Fms degradation induced by proteasome inhibitors was controlled by the activation of p38/tumor necrosis factor-alpha converting enzyme (TACE)-mediated regulated intramembrane proteolysis (RIPping). This was validated through the restoration of c-Fms using specific inhibitors of p38 and TACE, and a stimulation of p38-dependent TACE. In addition, c-Fms degradation by proteasome inhibition completely blocked M-CSF-mediated intrinsic signalling and led to the suppression of osteoclast differentiation and bone resorption. In a mouse model with intraperitoneal administration of lipopolysaccharide (LPS) that stimulates osteoclast formation and leads to bone loss, proteasome blockers prevented LPS-induced inflammatory bone resorption due to a decrease in the number of c-Fms-positive osteoclasts. Our study showed that accelerating c-Fms proteolysis by proteasome inhibitors may be a therapeutic option for inflammation-induced bone loss.