Showing papers on "MG132 published in 2018"

The polyphenol quercetin induces cell death in leukemia by targeting epigenetic regulators of pro-apoptotic genes

Abstract: In the present study, we investigated the molecular mechanisms underlying the pro-apoptotic effects of quercetin (Qu) by evaluating the effect of Qu treatment on DNA methylation and posttranslational histone modifications of genes related to the apoptosis pathway. This study was performed in vivo in two human xenograft acute myeloid leukemia (AML) models and in vitro using HL60 and U937 cell lines. Qu treatment almost eliminates DNMT1 and DNMT3a expression, and this regulation was in part STAT-3 dependent. The treatment also downregulated class I HDACs. Furthermore, treatment of the cell lines with the proteasome inhibitor, MG132, together with Qu prevented degradation of class I HDACs compared to cells treated with Qu alone, indicating increased proteasome degradation of class I HDACS by Qu. Qu induced demethylation of the pro-apoptotic BCL2L11, DAPK1 genes, in a dose- and time-dependent manner. Moreover, Qu (50 μmol/L) treatment of cell lines for 48 h caused accumulation of acetylated histone 3 and histone 4, resulting in three- to ten fold increases in the promoter region of DAPK1, BCL2L11, BAX, APAF1, BNIP3, and BNIP3L. In addition, Qu treatment significantly increased the mRNA levels of all these genes, when compared to cells treated with vehicle only (control cells) (*p < 0.05). In summary, our results showed that enhanced apoptosis, induced by Qu, might be caused in part by its DNA demethylating activity, by HDAC inhibition, and by the enrichment of H3ac and H4ac in the promoter regions of genes involved in the apoptosis pathway, leading to their transcription activation.

Leptin increases mitochondrial OPA1 via GSK3-mediated OMA1 ubiquitination to enhance therapeutic effects of mesenchymal stem cell transplantation.

Abstract: Accumulating evidence revealed that mesenchymal stem cells (MSCs) confer cardioprotection against myocardial infarction (MI). However, the poor survival and engraftment rate of the transplanted cells limited their therapeutic efficacy in the heart. The enhanced leptin production associated with hypoxia preconditioning contributed to the improved MSCs survival. Mitochondrial integrity determines the cellular fate. Thus, we aimed to investigate whether leptin can enhance mitochondrial integrity of human MSCs (hMSCs) to protect against various stress. In vivo, transplantation of leptin-overexpressing hMSCs into the infarcted heart resulted in improved cell viability, leading to enhanced angiogenesis and cardiac function. In vitro, pretreatment of hMSCs with recombinant leptin (hMSCs-Leppre) displayed improved cell survival against severe ischemic condition (glucose and serum deprivation under hypoxia), which was associated with increased mitochondrial fusion. Subsequently, Optic atrophy 1 (OPA1), a mitochondrial inner membrane protein that regulates fusion and cristae structure, was significantly elevated in the hMSCs-Leppre group, and the protection of leptin was abrogated by targeting OPA1 with a selective siRNA. Furthermore, OMA1, a mitochondrial protease that cleaves OPA1, decreased in a leptin-dependent manner. Pretreatment of cells with an inhibitor of the proteasome (MG132), prevented leptin-induced OMA1 degradation, implicating the ubiquitination/proteasome system as a part of the protective leptin pathway. In addition, GSK3 inhibitor (SB216763) was also involved in the degradation of OMA1. In conclusion, in the hostile microenvironment caused by MI, (a) leptin can maintain the mitochondrial integrity and prolong the survival of hMSCs; (b) leptin-mediated mitochondrial integrity requires phosphorylation of GSK3 as a prerequisite for ubiquitination-depended degradation of OMA1 and attenuation of long-OPA1 cleavage. Thus, leptin targeting the GSK3/OMA1/OPA1 signaling pathway can optimize hMSCs therapy for cardiovascular diseases such as MI.

Reduced Annexin A1 Secretion by ABCA1 Causes Retinal Inflammation and Ganglion Cell Apoptosis in a Murine Glaucoma Model.

Abstract: Variants near the ATP-binding cassette transporter A1 (ABCA1) gene are associated with elevated intraocular pressure and newly discovered risk factors for glaucoma. Previous studies have shown an association between ABCA1 deficiency and retinal inflammation. Using a mouse model of ischemia-reperfusion (IR) induced by acute intraocular pressure elevation, we found that the retinal expression of ABCA1 protein was decreased. An induction of ABCA1 expression by liver X receptor agonist TO901317 reduced retinal ganglion cell (RGC) apoptosis after IR and promoted membrane translocation and secretion of the anti-inflammatory factor annexin A1 (ANXA1). Moreover, ABCA1 and ANXA1 co-localized in cell membranes, and the interaction domain is amino acid 196 to 274 of ANXA1 fragment. TO901317 also reduced microglia migration and activation and decreased the expression of pro-inflammatory cytokines interleukin (IL)-17A and IL-1β, which could be reversed by the ANXA1 receptor blocker Boc2. Overexpression of TANK-binding kinase 1 (TBK1) increased ABCA1 degradation, which was reversed by the proteasome inhibitor carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132). Silencing Tbk1 with siRNA increased ABCA1 expression and promoted ANXA1 membrane translocation. These results indicate a novel IR mechanism, that leads via TBK1 activation to ABCA1 ubiquitination. This degradation decreases ANXA1 secretion, thus facilitating retinal inflammation and RGC apoptosis. Our findings suggest a potential treatment strategy to prevent RGC apoptosis in retinal ischemia and glaucoma.

TGF‐β inhibits osteogenesis by upregulating the expression of ubiquitin ligase SMURF1 via MAPK‐ERK signaling

Abstract: High incidence of osteoporotic fractures emphasizes the necessity of developing effective measures to promote osteogenesis. In our study, we investigated a possible role of MAPK-ERK signaling in the TGF-β-mediated osteoblastic differentiation. Our results indicated that TGF-β activated the MAPK-ERK pathway and inhibited osteogenesis in mesenchymal pluripotent cell line, C3H10T1/2, and preosteoblastic cell line, MC3T3 cells. And the downregulation of MAPK-ERK signaling using pharmacological inhibitor U0126 and RNA interference rescued osteoblast differentiation suppressed by TGF-β, which was confirmed by Alkaline phosphatase (ALP) staining and alizarrn red staining, and the enhanced expression of osteogenesic markers. Western blotting analysis indicated that TGF-β induced protein expression of E3 ubiquitin-protein ligase SMURF1, which contributed to the degradation of RUNX2 and SMAD1 as evidenced by SMURF1 inhibition using RNA interference and proteasome inhibitor MG132. Moreover, we observed that the expression of SMURF1 was decreased, while that of SMAD1 and RUNX2 increased by MAPK-ERK inhibitor U0126 in TGF-β-treated differentiating preosteoblasts, suggesting that MAPK-ERK regulated the transcription of osteogenesis-related genes. Furthermore, a synergistic effect between U0126 and bone morphogenic protein (BMP)-2 on osteoblast differentiation and bone formation was observed both in cell cultures and experimental animals. In conclusion, our results revealed that TGF-β inhibited osteoblastic differentiation by inducing the MAPK-ERK pathway which upregulated the expression of ubiquitin ligase SMURF1 and resulted in reduced presence of osteogenic proteins. In addition, the potentiation of BMP-2 on osteogenic activity by ERK1/2 inhibitor U0126 suggests that it may have potential clinical utility for promoting osteogenesis in bone fracture repair.

Zinc finger protein 746 promotes colorectal cancer progression via c-Myc stability mediated by glycogen synthase kinase 3β and F-box and WD repeat domain-containing 7.

Abstract: To elucidate the underlying oncogenic mechanism of zinc finger protein 746 (ZNF746), current study was conducted in colorectal cancers (CRCs). Herein, ZNF746 was overexpressed in HCT116, SW620, and SW480 cells, which was supported by CRC tissue microarray and TCGA analysis. Also, DNA microarray revealed the differentially expressed gene profile particularly related to cell cycle genes and c-Myc in ZNF746 depleted HCT116 cells. Furthermore, ZNF746 enhanced the stability of c-Myc via their direct binding through nuclear colocalization by immunoprecipitation and immunofluorescence, while ZNF746 and c-Myc exist mainly in nucleoplasm. Conversely, ZNF746 depletion attenuated phosphorylation of c-Myc (S62) and glycogen synthase kinase 3β (GSK3β) (S9) and also activated p-c-Myc (T58), which was reversed by GSK3 inhibitors such as SB-216763 and Enza. Also, c-Myc degradation by ZNF746 depletion was blocked by knockdown of F-box/WD repeat-containing protein 7 (FBW7) ubiquitin ligase or proteosomal inhibitor MG132. Additionally, the growth of ZNF746 depleted HCT116 cancer cells was retarded with decreased expression of ZNF746 and c-Myc. Overall, these findings suggest that ZNF746 promotes CRC progression via c-Myc stability mediated by GSK3 and FBW7.

Concurrent Inhibition of Pim and FLT3 Kinases Enhances Apoptosis of FLT3-ITD Acute Myeloid Leukemia Cells through Increased Mcl-1 Proteasomal Degradation

Abstract: Purpose:fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is present in 30% of acute myeloid leukemia (AML), and these patients have short disease-free survival. FLT3 inhibitors have limited and transient clinical activity, and concurrent treatment with inhibitors of parallel or downstream signaling may improve responses. The oncogenic serine/threonine kinase Pim-1 is upregulated downstream of FLT3-ITD and also promotes its signaling in a positive feedback loop, suggesting benefit of combined Pim and FLT3 inhibition.Experimental Design: Combinations of clinically active Pim and FLT3 inhibitors were studied in vitro and in vivoResults: Concurrent treatment with the pan-Pim inhibitor AZD1208 and FLT3 inhibitors at clinically applicable concentrations abrogated in vitro growth of FLT3-ITD, but not wild-type FLT3 (FLT3-WT), cell lines. AZD1208 cotreatment increased FLT3 inhibitor-induced apoptosis of FLT3-ITD, but not FLT3-WT, cells measured by sub-G1 fraction, annexin V labeling, mitochondrial membrane potential, and PARP and caspase-3 cleavage. Concurrent treatment with AZD1208 and the FLT3 inhibitor quizartinib decreased growth of MV4-11 cells, with FLT3-ITD, in mouse xenografts, and prolonged survival, enhanced apoptosis of FLT3-ITD primary AML blasts, but not FLT3-WT blasts or remission marrow cells, and decreased FLT3-ITD AML blast colony formation. Mechanistically, AZD1208 and quizartinib cotreatment decreased expression of the antiapoptotic protein Mcl-1. Decrease in Mcl-1 protein expression was abrogated by treatment with the proteasome inhibitor MG132, and was preceded by downregulation of the Mcl-1 deubiquitinase USP9X, a novel mechanism of Mcl-1 regulation in AML.Conclusions: The data support clinical testing of Pim and FLT3 inhibitor combination therapy for FLT3-ITD AML. Clin Cancer Res; 24(1); 234-47. ©2017 AACR.

Camptothecin induces G2/M phase arrest through the ATM-Chk2-Cdc25C axis as a result of autophagy-induced cytoprotection: Implications of reactive oxygen species.

Abstract: In the present study, we report that camptothecin (CPT) caused irreversible cell cycle arrest at the G2/M phase, and was associated with decreased levels of cell division cycle 25C (Cdc25C) and increased levels of cyclin B1, p21, and phospho-H3. Interestingly, the reactive oxygen species (ROS) inhibitor, glutathione, decreased CPT-induced G2/M phase arrest and moderately induced S phase arrest, indicating that the ROS is required for the regulation of CPT-induced G2/M phase arrest. Furthermore, transient knockdown of nuclear factor-erythroid 2-related factor 2 (Nrf2), in the presence of CPT, increased the ROS' level and further shifted the cell cycle from early S phase to the G2/M phase, indicating that Nrf2 delayed the S phase in response to CPT. We also found that CPT-induced G2/M phase arrest increased, along with the ataxia telangiectasia-mutated (ATM)-checkpoint kinase 2 (Chk2)-Cdc25C axis. Additionally, the proteasome inhibitor, MG132, restored the decrease in Cdc25C levels in response to CPT, and significantly downregulated CPT-induced G2/M phase arrest, suggesting that CPT enhances G2/M phase arrest through proteasome-mediated Cdc25C degradation. Our data also indicated that inhibition of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) inhibited CPT-induced p21 and cyclin B1 levels; however, inhibition of ERK blocked CPT-induced G2/M phase arrest, and inhibition of JNK enhanced apoptosis in response to CPT. Finally, we found that CPT-induced G2/M phase arrest circumvented apoptosis by activating autophagy through ATM activation. These findings suggest that CPT-induced G2/M phase arrest through the ROS-ATM-Chk2-Cdc25C axis is accompanied by the activation of autophagy.

Proteasome inhibitor bortezomib is a novel therapeutic agent for focal radiation-induced osteoporosis

Abstract: Bone atrophy and its related fragility fractures are frequent, late side effects of radiotherapy in cancer survivors and have a detrimental impact on their quality of life. In another study, we showed that parathyroid hormone 1-34 and anti-sclerostin antibody attenuates radiation-induced bone damage by accelerating DNA repair in osteoblasts. DNA damage responses are partially regulated by the ubiquitin proteasome pathway. In the current study, we examined whether proteasome inhibitors have similar bone-protective effects against radiation damage. MG132 treatment greatly reduced radiation-induced apoptosis in cultured osteoblastic cells. This survival effect was owing to accelerated DNA repair as revealed by γH2AX foci and comet assays and to the up-regulation of Ku70 and DNA-dependent protein kinase, catalytic subunit, essential DNA repair proteins in the nonhomologous end-joining pathway. Administration of bortezomib (Bzb) reversed the loss of trabecular bone structure and strength in mice at 4 wk after focal radiation. Histomorphometry revealed that Bzb significantly increased the number of osteoblasts and activity in the irradiated area and suppressed the number and activity of osteoclasts, regardless of irradiation. Two weeks of Bzb treatment accelerated DNA repair in bone-lining osteoblasts and thus promoted their survival. Meanwhile, it also inhibited bone marrow adiposity. Taken together, we demonstrate a novel role of proteasome inhibitors in treating radiation-induced osteoporosis.-Chandra, A., Wang, L., Young, T., Zhong, L., Tseng, W.-J., Levine, M. A., Cengel, K., Liu, X. S., Zhang, Y., Pignolo, R. J., Qin, L. Proteasome inhibitor bortezomib is a novel therapeutic agent for focal radiation-induced osteoporosis.

Hypoxia-inducible transgelin 2 selects epithelial-to-mesenchymal transition and γ-radiation-resistant subtypes by focal adhesion kinase-associated insulin-like growth factor 1 receptor activation in non-small-cell lung cancer cells.

Abstract: Microenvironment, such as hypoxia common to cancer, plays a critical role in the epithelial-to-mesenchymal transition (EMT) program, which is a major route of cancer metastasis and confers γ-radiation resistance to cells Herein, we showed that transgelin 2 (TAGLN2), an actin-binding protein, is significantly induced in hypoxic lung cancer cells and that Snail1 is simultaneously increased, which induces EMT by downregulating E-cadherin expression Forced TAGLN2 expression induced severe cell death; however, a small population of cells surviving after forced TAGLN2 overexpression showed γ-radiation resistance, which might promote tumor relapse and recurrence These surviving cells showed high metastatic activity with an increase of EMT markers including Snail1 In these cells, TAGLN2 activated the insulin-like growth factor 1 receptor β (IGF1Rβ)/PI3K/AKT pathway by recruitment of focal adhesion kinase to the IGF1R signaling complex Activation of the IGF1Rβ/PI3K/AKT pathway also induced inactivation of glycogen synthase kinase 3β (GSK3β), which is involved in Snail1 stabilization Therefore, both the IGF1Rβ inhibitor (AG1024) and the PI3K inhibitor (LY294002) or AKT inactivation with MK2206 lower the cellular level of Snail1 Involvement of GSK3β was also confirmed by treatment with lithium chloride, the inducer of GSK3β phosphorylation, or MG132, the 26S proteasomal inhibitor, which also stabilized Snail1 In conclusion, the present study provides important evidence that hypoxia-inducible TAGLN2 is involved in the selection of cancer cells with enhanced EMT properties to overcome the detrimental environment of cancer cells

ERK is a negative feedback regulator for IFN-γ/STAT1 signaling by promoting STAT1 ubiquitination.

Abstract: We recently reported that STAT1 plays a tumor suppressor role, and ERK was inversely correlation with STAT1 expression in esophageal squamous cell carcinoma (ESCC). Here, we investigated the mechanism(s) that are responsible for the ERK regulates STAT1 in ESCC. We performed the immunoprecipitation (IP) to detect the ubiquitin of STAT1 upon MEK transfection or U0126 treatment and co-IP to confirm the binding of STAT1 and ERK in ESCC cell lines. We found evidence that the ubiquitin-proteasome pathway can efficiently degrade STAT1 in ESCC cells, as MG132 treatment rapidly and dramatically increased STAT1 expression in these cells. This process is not dependent on the phosphorylation of the two important STAT1 residues, Y701 and S727, as site-directed mutagenesis of these two sites did not affect STAT1 degradation. We also found that ERK promotes proteasome degradation of STAT1, supported by the observations that pharmacologic inhibition of ERK resulted in a substantial increase of STAT1 whereas expression of constitutively active ERK further reduced the STAT1 protein level. In addition to suppressing STAT1 expression, ERK limited STAT1 signaling by decreasing the production of IFNγ. To conclude, ERK is an effective negative regulator of STAT1 signaling in ESCC, by promoting its proteasome degradation and decreasing IFNγ production. Our data further supports that targeting ERK and/or STAT1 may be useful for treating ESCC.

Autophagy Stimulus Promotes Early HuR Protein Activation and p62/SQSTM1 Protein Synthesis in ARPE-19 Cells by Triggering Erk1/2, p38MAPK, and JNK Kinase Pathways.

Abstract: RNA-binding protein dysregulation and altered expression of proteins involved in the autophagy/proteasome pathway play a role in many neurodegenerative disease onset/progression, including age-related macular degeneration (AMD). HuR/ELAVL1 is a master regulator of gene expression in human physiopathology. In ARPE-19 cells exposed to the proteasomal inhibitor MG132, HuR positively affects at posttranscriptional level p62 expression, a stress response gene involved in protein aggregate clearance with a role in AMD. Here, we studied the early effects of the proautophagy AICAR + MG132 cotreatment on the HuR-p62 pathway. We treated ARPE-19 cells with Erk1/2, AMPK, p38MAPK, PKC, and JNK kinase inhibitors in the presence of AICAR + MG132 and evaluated HuR localization/phosphorylation and p62 expression. Two-hour AICAR + MG132 induces both HuR cytoplasmic translocation and threonine phosphorylation via the Erk1/2 pathway. In these conditions, p62 mRNA is loaded on polysomes and its translation in de novo protein is favored. Additionally, for the first time, we report that JNK can phosphorylate HuR, however, without modulating its localization. Our study supports HuR’s role as an upstream regulator of p62 expression in ARPE-19 cells, helps to understand better the early events in response to a proautophagy stimulus, and suggests that modulation of the autophagy-regulating kinases as potential therapeutic targets for AMD may be relevant.

Neotuberostemonine inhibits the differentiation of lung fibroblasts into myofibroblasts in mice by regulating HIF-1α signaling.

Abstract: Pulmonary fibrosis may be partially the result of deregulated tissue repair in response to chronic hypoxia. In this study we explored the effects of hypoxia on lung fibroblasts and the effects of neotuberostemonine (NTS), a natural alkaloid isolated from Stemona tuberosa, on activation of fibroblasts in vitro and in vivo. PLFs (primary mouse lung fibroblasts) were activated and differentiated after exposure to 1% O2 or treatment with CoCl2 (100 μmol/L), evidenced by markedly increased protein or mRNA expression of HIF-1α, TGF-β, FGF2, α-SMA and Col-1α/3α, which was blocked after silencing HIF-1α, suggesting that the activation of fibroblasts was HIF-1α-dependent. NTS (0.1–10 μmol/L) dose-dependently suppressed hypoxia-induced activation and differentiation of PLFs, whereas the inhibitory effect of NTS was abolished by co-treatment with MG132, a proteasome inhibitor. Since prolyl hydroxylation is a critical step in initiation of HIF-1α degradation, we further showed that NTS treatment reversed hypoxia- or CoCl2-induced reduction in expression of prolyl hydroxylated-HIF-1α. With hypoxyprobe immunofiuorescence staining, we showed that NTS treatment directly reversed the lower oxygen tension in hypoxia-exposed PLFs. In a mouse model of lung fibrosis, oral administration of NTS (30 mg·kg-1·d-1, for 1 or 2 weeks) effectively attenuated bleomycin-induced pulmonary fibrosis by inhibiting the levels of HIF-1α and its downstream profibrotic factors (TGF-β, FGF2 and α-SMA). Taken together, these results demonstrate that NTS inhibits the protein expression of HIF-1α and its downstream factors TGF-β, FGF2 and α-SMA both in hypoxia-exposed fibroblasts and in lung tissues of BLM-treated mice. NTS with anti-HIF-1α activity may be a promising pharmacological agent for the treatment of pulmonary fibrosis.

Transcriptional regulation of Runx2 by HSP90 controls osteosarcoma apoptosis via the AKT/GSK-3β/β-catenin signaling.

Abstract: Osteosarcoma (OS) is the most malignant primary bone tumor in children and adolescents with limited treatment options and poor prognosis. Recently, aberrant expression of Runx2 has been found in OS, thereby contributing to the development, and progression of OS. However, the upstream signaling molecules that regulate its expression in OS remain largely unknown. In the present study, we first confirmed that the inhibition of HSP90 with 17-AAG caused significant apoptosis of OS cells via a caspase-3-dependent mechanism, and that inhibition or knockdown of HSP90 by 17-AAG or siRNAs significantly suppressed mRNA and protein expression of Runx2. Furthermore, we provided evidence that Runx2 was transcriptionally regulated by HSP90 when using MG132 and CHX chase assay. We also demonstrated that β-catenin was overexpressed in OS tissue, and that knockdown of β-catenin induced pronounced apoptosis of OS cells in the presence or absence of 17-AAG. Interestingly, this phenomenon was accompanied with a significant reduction of Runx2 and Cyclin D1 expression, indicating an essential role of Runx2/Cyclin D1 in 17-AAG-induced cells apoptosis. Moreover, we demonstrated that the apoptosis of OS cells induced by 17-AAG did require the involvement of the AKT/GSK-3β/β-catenin signaling pathway by using pharmacological inhibitor GSK-3β (LiCl) or siGSK-3β. Our findings reveal a novel mechanism that Runx2 is transcriptionally regulated by HSP90 via the AKT/GSK-3β/β-catenin signaling pathway, and by which leads to apoptosis of OS cells.

Angiotensin II Promotes KV7.4 Channels Degradation Through Reduced Interaction With HSP90 (Heat Shock Protein 90)

Abstract: Voltage-gated Kv7.4 channels have been implicated in vascular smooth muscle cells' activity because they modulate basal arterial contractility, mediate responses to endogenous vasorelaxants, and are downregulated in several arterial beds in different models of hypertension. Angiotensin II (Ang II) is a key player in hypertension that affects the expression of several classes of ion channels. In this study, we evaluated the effects of Ang II on the expression and function of vascular Kv7.4. Western blot and quantitative polymerase chain reaction revealed that in whole rat mesenteric artery, Ang II incubation for 1 to 7 hours decreased Kv7.4 protein expression without reducing transcript levels. Moreover, Ang II decreased XE991 (Kv7)-sensitive currents and attenuated membrane potential hyperpolarization and relaxation induced by the Kv7 activator ML213. Ang II also reduced Kv7.4 staining at the plasma membrane of vascular smooth muscle cells. Proteasome inhibition with MG132 prevented Ang II-induced decrease of Kv7.4 levels and counteracted the functional impairment of ML213-induced relaxation in myography experiments. Proximity ligation assays showed that Ang II impaired the interaction of Kv7.4 with the molecular chaperone HSP90 (heat shock protein 90), enhanced the interaction of Kv7.4 with the E3 ubiquitin ligase CHIP (C terminus of Hsp70-interacting protein), and increased Kv7.4 ubiquitination. Similar alterations were found in mesenteric vascular smooth muscle cells isolated from Ang II-infused mice. The effect of Ang II was emulated by 17-AAG (17-demethoxy-17-(2-propenylamino) geldanamycin) that inhibits HSP90 interactions with client proteins. These results show that Ang II downregulates Kv7.4 by altering protein stability through a decrease of its interaction with HSP90. This leads to the recruitment of CHIP and Kv7.4 ubiquitination and degradation via the proteasome.

Roasted Coffee Reduces β-Amyloid Production by Increasing Proteasomal β-Secretase Degradation in Human Neuroblastoma SH-SY5Y Cells

Abstract: Scope Epidemiological studies have shown that coffee consumption may be associated with a lower risk of developing several neurological disorders, including Alzheimer's disease (AD). Caffeine is a prominent candidate component underlying the preventive effects of coffee; however, the contribution of other constituents is unclear. To clarify this issue, the effect of roasting coffee beans on β-secretase (BACE1) expression in human neuroblastoma SH-SY5Y cells is investigated. Methods and results Coffee (2%) reduces Aβ accumulation in culture medium to 80% of control levels after 24 h. Accordingly, BACE1 expression is decreased to 70% of control levels at 12 h. Experiments using cycloheximide and MG132, a proteasome inhibitor, reveal that coffee enhanced BACE1 degradation through activation of proteasomal activity. Furthermore, coffee activates cAMP-dependent protein kinase, and consequently, phosphorylation of a serine residue of proteasome 26S subunit, non-ATPase 11 (PSMD11). Pyrocatechol, a strong antioxidant known as catechol or 1,2-dihydroxybenzene, produced from chlorogenic acid during roasting, also reduces BACE1 expression by activation of proteasomal activity. Furthermore, pyrocatechol reduces Aβ production in SH-SY5Y cells. Conclusion The data suggest that the roasting process may be crucial for the protective effects of coffee consumption in AD.

Endoplasmic reticulum-associated degradation and disposal of misfolded GPI-anchored proteins in Trypanosoma brucei.

Abstract: Misfolded secretory proteins are retained by endoplasmic reticulum quality control (ERQC) and degraded in the proteasome by ER-associated degradation (ERAD). However, in yeast and mammals, misfolded glycosylphosphatidylinositol (GPI)-anchored proteins are preferentially degraded in the vacuole/lysosome. We investigate this process in the divergent eukaryotic pathogen Trypanosoma brucei using a misfolded GPI-anchored subunit (HA:E6) of the trypanosome transferrin receptor. HA:E6 is N-glycosylated and GPI-anchored and accumulates in the ER as aggregates. Treatment with MG132, a proteasome inhibitor, generates a smaller protected polypeptide (HA:E6*), consistent with turnover in the proteasome. HA:E6* partitions between membrane and cytosol fractions, and both pools are proteinase K-sensitive, indicating cytosolic disposition of membrane-associated HA:E6*. HA:E6* is de-N-glycosylated and has a full GPI-glycan structure from which dimyristoylglycerol has been removed, indicating that complete GPI removal is not a prerequisite for proteasomal degradation. However, HA:E6* is apparently not ubiquitin-modified. The trypanosome GPI anchor is a forward trafficking signal; thus the dynamic tension between ERQC and ER exit favors degradation by ERAD. These results differ markedly from the standard eukaryotic model systems and may indicate an evolutionary advantage related to pathogenesis.

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.

Abstract: 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.

Inhibitors of the proteasome stimulate the epithelial sodium channel (ENaC) through SGK1 and mimic the effect of aldosterone

Abstract: The epithelial sodium channel (ENaC) marks the tightly regulated, rate-limiting step of sodium re-absorption in the aldosterone-sensitive distal nephron (ASDN). Stimulation of ENaC activity by aldosterone involves the serum and glucocorticoid-induced kinase 1 (SGK1) and is mediated via complex mechanisms including inhibition of channel retrieval. Retrieved channels may be recycled or degraded, e.g. by the proteasomal pathway. The aim of the present study was to investigate whether inhibitors of the proteasome affect ENaC activity and surface expression, and to explore a possible involvement of SGK1. Short circuit current (I SC) measurements were performed on confluent mCCDcl1 murine cortical collecting duct cells to investigate the effect of two distinct proteasomal inhibitors, MG132 and bortezomib, on amiloride-sensitive ENaC-mediated I SC. Both inhibitors robustly stimulated amiloride-sensitive I SC. The time course and magnitude of the stimulatory effect of the proteasomal inhibitors on I SC were similar to those of aldosterone. Both, MG132 and aldosterone, significantly increased the abundance of β-ENaC at the cell surface. SGK1 activity was assessed by monitoring the phosphorylation of a downstream target, NDRG1, and was found to be increased by MG132. Importantly, inhibiting SGK1 activity prevented not only the stimulatory effect of aldosterone but also that of proteasomal inhibition. In conclusion, these data suggest that ENaC stimulation following proteasomal inhibition is due to an accumulation of active SGK1 resulting in increased expression of ENaC at the cell surface. Thus, inhibition of the proteasome mimics SGK1-dependent stimulation of ENaC by aldosterone.

Quantitative peptidomics of endogenous peptides involved in TGF-β1-induced epithelial mesenchymal transition of renal epithelial cells.

Abstract: TGF-β1 is a key fibrotic factor mediating epithelial mesenchymal transition (EMT) of epithelial cells through various signaling pathways. However, roles of proteolytic cleavage and endogenous peptide dynamics in TGF-β1-induced EMT remain unknown. We therefore performed quantitative peptidomics of TGF-β1-induced EMT in renal tubular epithelial cells. The acquired mesenchymal characteristics were confirmed, including morphological change (from cobblestone-like to fibroblast-like), decreased epithelial marker (ZO-1), and increased mesenchymal marker (vimentin). Quantitative peptidomics using stable isotope labeling revealed significantly altered levels of 70 unique endogenous peptides (derived from internal and C-terminal parts of 39 unique precursor proteins) after EMT induction. Interestingly, the majority of these peptides were derived from non-short-lived proteins, and analysis of P1 position revealed predominance of hydrophobic residues, suggesting that these endogenous peptides were generated mainly from proteasome cleavage. This hypothesis was confirmed by treating the cells with MG132 (a proteasome inhibitor), which provided almost identical endogenous peptide pattern as of the TGF-β1-treated cells. Moreover, validation assay showed marked reduction of proteasome peptidase activity in both TGF-β1-treated and MG132-treated cells. This is the first peptidome dataset that provides several novel aspects of mechanisms for TGF-β1-induced EMT. Our data also suggest that TGF-β1 exerts inhibitory effect against proteasome activity during EMT induction.

GRWD1 regulates ribosomal protein L23 levels via the ubiquitin-proteasome system.

Abstract: Glutamate-rich WD40 repeat-containing 1 (GRWD1) is a Cdt1-binding protein that promotes mini-chromosome maintenance (MCM) loading through its histone chaperone activity. GRWD1 acts as a tumor-promoting factor by downregulating p53 (also known as TP53) via the RPL11-MDM2-p53 axis. Here, we identified GRWD1-interacting proteins using a proteomics approach and showed that GRWD1 interacts with various proteins involved in transcription, translation, DNA replication and repair, chromatin organization, and ubiquitin-mediated proteolysis. We focused on the ribosomal protein ribosomal protein L23 (RPL23), which positively regulates nucleolar stress responses through MDM2 binding and inhibition, thereby functioning as a tumor suppressor. Overexpression of GRWD1 decreased RPL23 protein levels and stability; this effect was restored upon treatment with the proteasome inhibitor MG132. EDD (also known as UBR5), an E3 ubiquitin ligase that interacts with GRWD1, also downregulated RPL23, and the decrease was further enhanced by co-expression of GRWD1. Conversely, siRNA-mediated GRWD1 knockdown upregulated RPL23. Co-expression of GRWD1 and EDD promoted RPL23 ubiquitylation. These data suggest that GRWD1 acts together with EDD to negatively regulate RPL23 via the ubiquitin-proteasome system. GRWD1 expression reversed the RPL23-mediated inhibition of anchorage-independent growth in cancer cells. Our data suggest that GRWD1-induced RPL23 proteolysis plays a role in downregulation of p53 and tumorigenesis.

Sulforaphane attenuates postnatal proteasome inhibition and improves spatial learning in adult mice.

Abstract: Proteasomes are known to degrade proteins involved in various processes like metabolism, signal transduction, cell-cycle regulation, inflammation, and apoptosis. Evidence showed that protein degradation has a strong influence on developing neurons as well as synaptic plasticity. Here, we have shown that sulforaphane (SFN) could prevent the deleterious effects of postnatal proteasomal inhibition on spatial reference and working memory of adult mice. One day old Balb/c mice received intracerebroventricular injections of MG132 and SFN. Sham received an equal volume of aCSF. We observed that SFN pre-administration could attenuate MG132 mediated decrease in proteasome and calpain activities. In vitro findings revealed that SFN could induce proteasomal activity by enhancing the expression of catalytic subunit-β5. SFN pre-administration prevented the hippocampus based spatial memory impairments during adulthood, mediated by postnatal MG132 exposure. Histological examination showed deleterious effects of MG132 on pyramidal neurons and granule cell neurons in DG and CA3 sub-regions respectively. Furthermore, SFN pre-administration has shown to attenuate the effect of MG132 on proteasome subunit-β5 expression and also induce the Nrf2 nuclear translocation. In addition, SFN pre-administered mice have also shown to induce expression of pCaMKII, pCreb, and mature/pro-Bdnf, molecules which play a crucial role in spatial learning and memory consolidation. Our findings have shown that proteasomes play an important role in hippocampal synaptic plasticity during the early postnatal period and SFN pre-administration could enhance the proteasomal activity as well as improve spatial learning and memory consolidation.

MG132 selectively upregulates MICB through the DNA damage response pathway in A549 cells.

Abstract: Natural killer (NK) cells recognize stress‑activated NK group 2, member D (NKG2D) ligands in tumors. In the present study, the expression levels of NKG2D ligands were examined in four lung cancer cell lines (A549, PLA801D, NCI‑H157 and NCI‑H520). In the A549 cells, the expression of MHC class I polypeptiderelated sequence (MIC)A/B and UL16 binding protein (ULBP)1 was weak, the expression of ULBP2 was typical, and neither ULBP3 nor ULBP4 were expressed. The mechanism underlying the regulatory effect of a cancer treatment agent on the expression of NKG2D ligands was investigated using the proteasome inhibitor MG132. Following treatment for 8 h with MG132, the transcription levels of MICB and ULBP1 were upregulated 10.62‑ and 11.09‑fold, respectively, and the expression levels of MICB and ULBP1 were increased by 68.18 and 23.65%, respectively. Notably, MICB exhibited significant time‑dependent change. MG132 increased the transcription of MICB by acting at a site in the 480‑bp MICB upstream promoter. The activity of the MICB promoter was upregulated 1.77‑fold following treatment with MG132. MG132 treatment improved the cytotoxicity of NK cells, which was partially blocked by an antibody targeting NKG2D, and more specifically the MICB molecule. The expression of MICB induced by MG132 was inhibited by KU‑55933 [ataxia telangiectasia mutated (ATM) kinase inhibitor], wortmannin (phosphoinositide 3 kinase inhibitor) and caffeine (ATM/ATM‑Rad3‑related inhibitor). The phosphorylation of checkpoint kinase 2 (Chk2), an event associated with DNA damage, was observed following treatment with MG132. These results indicated that MG132 selectively upregulates the expression of MICB in A549 cells, and increases the NKG2D‑mediated cytotoxicity of NK cells. The regulatory effect of MG132 may be associated with the activation of Chk2, an event associated with DNA damage. The combination of MG132 with NK cell immunotherapy may have a synergistic effect that improves the therapeutic effect of lung cancer treatment.

ING5-mediated antineuroblastoma effects of suberoylanilide hydroxamic acid.

Abstract: Neuroblastoma is the most common extracranial solid neuroendocrine cancer and is one of the leading causes of death in children. To improve clinical outcomes and prognosis, discovering new promising drugs and targeted medicine is essential. We found that applying Suberoylanilide hydroxamic acid (SAHA; Vorinostat, a histone deacetylase inhibitor) and MG132 (a proteasome inhibitor) to SH-SY5Y cells synergistically suppressed proliferation, glucose metabolism, migration, and invasion and induced apoptosis and cell cycle arrest. These effects occurred both concentration and time dependently and were associated with the effects observed with inhibitor of growth 5 (ING5) overexpression. SAHA and MG132 treatment increased the expression levels of ING5, PTEN, p53, Caspase-3, Bax, p21, and p27 but decreased the expression levels of 14-3-3, MMP-2, MMP-9, ADFP, Nanog, c-myc, CyclinD1, CyclinB1, and Cdc25c concentration dependently, similar to ING5. SAHA may downregulate miR-543 and miR-196-b expression to enhance the translation of ING5 protein, which promotes acetylation of histones H3 and H4. All three proteins (ING5 and acetylated histones H3 and H4) were recruited to the promoters of c-myc, Nanog, CyclinD1, p21, and p27 for complex formation, thereby regulating the mRNA expression of downstream genes. ING5 overexpression and SAHA and/or MG132 administration inhibited tumor growth in SH-SY5Y cells by suppressing proliferation and inducing apoptosis. The expression of acetylated histones H3 and ING5 may be closely linked to the tumor size of neuroblastomas. In summary, SAHA and/or MG132 can synergistically suppress the malignant phenotypes of neuroblastoma cells through the miRNA-ING5-histone acetylation axis and via proteasomal degradation, respectively. Therefore, the two drugs may serve as potential treatments for neuroblastoma.

Ubiquitin-Proteasome System Is Required for Efficient Replication of Singapore Grouper Iridovirus.

Abstract: The ubiquitin-proteasome system (UPS) serves as the major intracellular pathway for protein degradation and plays crucial roles in several cellular processes. However, little is known about the potential actions of the UPS during fish virus infection. In this study, we elucidated the possible roles of UPS in the life cycle of Singapore grouper iridovirus (SGIV); a large DNA virus that usually causes serious systemic diseases with high mortality in groupers. Data from transcriptomic analysis of differentially expressed genes illustrated that expression of 65 genes within the UPS pathway, including ubiquitin encoding, ubiquitination, deubiquitination, and proteasome, were up- or down-regulated during SGIV infection. Using different proteasome inhibitors, inhibition of the proteasome decreased SGIV replication in vitro, accompanied by inhibition of virus assembly site formation, and viral gene transcription and protein transportation. Over-expression of ubiquitin partly rescued the inhibitory effect of ubiquitin inhibitor on SGIV replication, suggesting that UPS was required for fish iridovirus infection in vitro. Viral or host proteins regulated by proteasome inhibition during SGIV infection were investigated with two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Sixty-two differentially expressed proteins, including 15 viral and 47 host proteins, were identified after SGIV infection. The host proteins were involved in ubiquitin-mediated protein degradation, metabolism, cytoskeleton, macromolecular biosynthesis, and signal transduction. Among them, 11 proteins were negatively regulated upon MG132 treatment during SGIV infection. This is believed to be the first study to provide evidence that UPS was essential for fish virus infection and replication.