scholarly journals Exogenous Therapeutics of Microrna-29a Attenuates Development of Hepatic Fibrosis in Cholestatic Animal Model through Regulation of Phosphoinositide 3-Kinase p85 Alpha

2020 ◽  
Vol 21 (10) ◽  
pp. 3636 ◽  
Author(s):  
Ya-Ling Yang ◽  
Feng-Sheng Wang ◽  
Hung-Yu Lin ◽  
Ying-Hsien Huang
Keyword(s):  
Liver Fibrosis ◽  
Unfolded Protein Response ◽  
Luciferase Reporter ◽  
Heat Shock Protein 60 ◽  
Unfolded Protein ◽  
Duct Ligation ◽  
Phosphoinositide 3 Kinase ◽  
Human Liver Cirrhosis ◽  
Protein Response

Recent studies have found that microRNA-29a (miR-29a) levels are significantly lower in fibrotic livers, as shown with human liver cirrhosis. Such downregulation influences the activation of hepatic stellate cells (HSC). Phosphoinositide 3-kinase p85 alpha (PI3KP85α) is implicated in the regulation of proteostasis mitochondrial integrity and unfolded protein response (UPR) and apoptosis in hepatocytes. This study aimed to investigate the potential therapeutic role of miR-29a in a murine bile duct ligation (BDL)-cholestatic injury and liver fibrosis model. Mice were assigned to four groups: sham, BDL, BDL + scramble miRs, and BDL + miR-29a-mimic. Liver fibrosis and inflammation were assessed by histological staining and mRNA/protein expression of representative markers. Exogenous therapeutics of miR-29a in BDL-stressed mice significantly attenuated glutamic oxaloacetic transaminase (GOT)/glutamic-pyruvic transaminase (GPT) and liver fibrosis, and caused a significant downregulation in markers related to inflammation (IL-1β), fibrogenesis (TGF-β1, α-SMA, and COL1α1), autophagy (p62 and LC3B II), mitochondrial unfolded protein response (UPRmt; C/EBP homologous protein (CHOP), heat shock protein 60 (HSP60), and Lon protease-1 (LONP1, a mitochondrial protease), and PI3KP85α within the liver tissue. An in vitro luciferase reporter assay further confirmed that miR-29a mimic directly targets mRNA 3′ untranslated region (UTR) of PI3KP85α to suppress its expression in HepG2 cell line. Our data provide new insights that therapeutic miR-29a improves cholestasis-induced hepatic inflammation and fibrosis and proteotstasis via blocking PI3KP85α, highlighting the potential of miR-29a targeted therapy for liver injury.

scholarly journals USP19 deubiquitinating enzyme inhibits muscle cell differentiation by suppressing unfolded-protein response signaling

2015 ◽  
Vol 26 (5) ◽  
pp. 913-923 ◽  
Author(s):  
Benjamin Wiles ◽  
Miao Miao ◽  
Erin Coyne ◽  
Louise Larose ◽  
Andrey V. Cybulsky ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Unfolded Protein Response ◽  
Muscle Cell ◽  
Deubiquitinating Enzyme ◽  
Muscle Cell Differentiation ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

USP19 deubiquitinating enzyme has two isoforms, cytoplasmic and endoplasmic reticulum (ER) localized. The ER-localized isoform specifically suppresses muscle cell differentiation in vitro and appears to do so by inhibiting the unfolded-protein response that occurs during such differentiation. In vivo, loss of USP19 promotes muscle regeneration following injury.

scholarly journals Mitochondrial Activity and Unfolded Protein Response are Required for Neutrophil Differentiation

2018 ◽  
Vol 47 (5) ◽  
pp. 1936-1950 ◽  
Author(s):  
Ayako Tanimura ◽  
Keiko Miyoshi ◽  
Taigo Horiguchi ◽  
Hiroko Hagita ◽  
Koichi Fujisawa ◽  
...  
Keyword(s):  
Er Stress ◽  
Unfolded Protein Response ◽  
Morphological Differentiation ◽  
Hematopoietic Differentiation ◽  
Macrophage Differentiation ◽  
Mitochondrial Energy Metabolism ◽  
Unfolded Protein ◽  
Protein Response ◽  
Neutrophil Differentiation

Background/Aims: Endoplasmic reticulum (ER) stress and unfolded protein response (UPR) are involved in hematopoietic differentiation. However, the mechanistic linkage between ER stress/UPR and hematopoietic differentiation remains unclear. Methods: We used bipotent HL-60 cells as an in vitro hematopoietic differentiation system to investigate the role of ER stress and UPR activity in neutrophil and macrophage differentiation. Results: The in vitro differentiation analysis revealed that ER stress decreased during both neutrophil and macrophage differentiations, and the activities of PERK and ATF6 were decreased and that of IRE1α was increased during neutrophil differentiation in a stage-specific manner. By contrast, the activities of ATF6 and ATF4 decreased during macrophage differentiation. When the cells were treated with oligomycin, the expression of CD11b, a myelocytic differentiation marker, and morphological differentiation were suppressed, and XBP-1 activation was inhibited during neutrophil differentiation, whereas CD11b expression was maintained, and morphological differentiation was not obviously affected during macrophage differentiation. Conclusion: In this study, we demonstrated that neutrophil differentiation is regulated by ER stress/UPR that is supported by mitochondrial ATP supply, in which IRE1α-XBP1 activation is essential. Our findings provide the evidence that mitochondrial energy metabolism may play a critical role in neutrophil differentiation.

scholarly journals Knockdown of cytokeratin 8 overcomes chemoresistance of chordoma cells by aggravating endoplasmic reticulum stress through PERK/eIF2α arm of unfolded protein response and blocking autophagy

2019 ◽  
Vol 10 (12) ◽  
Author(s):  
Di Wang ◽  
Peiran Zhang ◽  
Xiaolong Xu ◽  
Jianhui Wang ◽  
Dong Wang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
Unfolded Protein Response ◽  
Xenograft Model ◽  
Spinal Tumor ◽  
Systematic Investigation ◽  
Tumor Xenograft ◽  
Unfolded Protein ◽  
Protein Response

AbstractChordoma is a malignant primary osseous spinal tumor with pronounced chemoresistance. However, the mechanisms of how chordoma cells develop chemoresistance are still not fully understood. Cytokeratin 8 (KRT8) is a molecular marker of notochordal cells, from which chordoma cells were believed to be originated. In this study, we showed that either doxorubicin or irinotecan promoted KRT8 expression in both CM319 and UCH1 cell lines, accompanied by an increased unfolded protein response and autophagy activity. Then, siRNA-mediated knockdown of KRT8 chemosensitized chordoma cells in vitro. Mechanistic studies showed that knockdown of KRT8 followed by chemotherapy aggravated endoplasmic reticulum stress through PERK/eIF2α arm of unfolded protein response and blocked late-stage autophagy. Moreover, suppression of the PERK/eIF2α arm of unfolded protein response using PERK inhibitor GSK2606414 partially rescued the apoptotic chordoma cells but did not reverse the blockage of the autophagy flux. Finally, tumor xenograft model further confirmed the chemosensitizing effects of siKRT8. This study represents the first systematic investigation into the role of KRT8 in chemoresistance of chordoma and our results highlight a possible strategy of targeting KRT8 to overcome chordoma chemoresistance.

scholarly journals Stimulation of surface IgM of chronic lymphocytic leukemia cells induces an unfolded protein response dependent on BTK and SYK

Blood ◽  
2014 ◽  
Vol 124 (20) ◽  
pp. 3101-3109 ◽  
Author(s):  
Sergey Krysov ◽  
Andrew J. Steele ◽  
Vania Coelho ◽  
Adam Linley ◽  
Marina Sanchez Hidalgo ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Unfolded Protein Response ◽  
Cell Receptor ◽  
Immunoglobulin M ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Unfolded Protein ◽  
Protein Response ◽  
Stimulation Of

Key Points Stimulation of the B-cell receptor of chronic lymphocytic leukemia cells results in activation of an unfolded protein response. Unfolded protein response activation following surface immunoglobulin M stimulation in vitro is dependent on the activity of BTK and SYK.

scholarly journals Activation of the Unfolded Protein Response with the First-in-Class P97 Inhibitor CB-5083 Induces Stable Disease Regression and Overcomes Ara-C Resistance in AML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1350-1350
Author(s):  
Steffan T. Nawrocki ◽  
Yingchun Han ◽  
Ronan LE Moigne ◽  
Valeria Visconte ◽  
Bartlomiej Przychodzen ◽  
...  
Keyword(s):  
Unfolded Protein Response ◽  
Board Of Directors ◽  
Cell Lines ◽  
Primary Cells ◽  
Advisory Committees ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

Abstract Acute myeloid leukemia (AML) therapy has remained relatively unchanged for more than 40 years with the majority of patients not achieving long-term remission when treated with currently available agents. Novel strategies are urgently needed to improve outcomes. The constitutive dysregulation of protein synthesis/turnover contributes to disease progression and drug resistance in many forms of cancer including AML. p97 (VCP) is a master regulator of protein turnover that has been implicated in oncogenesis and malignant pathogenesis. CB-5083 is a first-in-class selective and potent orally available inhibitor of p97 that in currently being evaluated in phase I clinical trials in patients with multiple myeloma and advanced solid tumors. To assess the potential benefit of p97 inhibition as a novel approach for AML therapy, we investigated the efficacy, pharmacodynamics (PD), and pharmacokinetics (PK) of CB-5083 in a panel of human AML cell lines with diverse genetic backgrounds, primary AML specimens from both newly diagnosed and relapsed/refractory patients, and xenograft mouse models of AML. In vitro treatment with CB-5083 potently diminished the viability of AML cell lines (n = 7) and primary CD34+ blasts obtained from patients (n = 10) with IC50s significantly below 1 µM (range 200 - 700 nM) in all lines and specimens evaluated to date. Diminished viability was associated with reduced clonogenic survival and increased apoptosis in AML cell lines and primary blasts. In contrast to many conventional and experimental drugs that are less active against primary AML cells than established AML cell lines, primary cells exhibited sensitivity to CB-5083 that was similar to cell lines. Additionally, CB-5083 was highly active in 3 different cell line models of cytarabine resistance and primary cells from refractory AML patients. This suggests that CB-5083 may be effective for patients who are relapsed/refractory to conventional therapy. In vitro PD analyses demonstrated that CB-5083 rapidly triggered the accumulation of ubiquitin-conjugated proteins, activated the unfolded protein response (UPR), disrupted STAT5 signaling, reduced levels of key STAT5 targets including BCL-xL and PIM-2, and induced apoptosis. The pro-apoptotic effects of CB-5083 were associated with activation of the endoplasmic reticulum (ER) resident initiator caspase-4 and induction of the BH3-only protein NOXA, which has been previously demonstrated to be an important mediator of cell death induced by other agents that disrupt protein homeostasis. RNA sequencing (RNASeq) gene ontology (GO) analyses of MV4-11 and MOLM-13 AML cells following treatment with CB-5083 demonstrated that short-term treatment (6h) caused significant increases in multiple regulators of the unfolded protein response, protein biosynthesis, and other ubiquitin-related pathways (p<0.001). Results were confirmed by qRT-PCR. The in vivo anti-leukemic activity of CB-5083 was investigated in two different xenograft mouse models of AML: the FLT3-ITD+ MV4-11 cell line and APML HL-60 cells. Oral administration of CB-5083 (once daily, 4 days on, 3 days off) was well tolerated and induced disease regression in both xenograft models (p<0.01). In vivo PD studies demonstrated that administration of CB-5083 led to reduced AML cell proliferation (PCNA), to the induction of apoptosis (active caspase-3), and pathway inhibition as evidenced by poly-ubiquitin accumulation and elevated expression of CHOP, GRP78, and NOXA. PK-PD analyses demonstrated a correlation between the kinetics of the in vivo PD effects and drug exposure. Our collective preclinical data demonstrate that p97 inhibition is a very effective novel anti-leukemic strategy and support clinical investigation of CB-5083 in patients with relapsed/refractory AML. Disclosures LE Moigne: Cleave Biosciences: Employment. Rolfe:Cleave Biosciences: Employment. Djakovic:Cleave Biosciences: Employment. Anderson:Cleave Biosciences: Employment. Wustrow:Cleave Biosciences: Employment. Zhou:Cleave Biosciences: Employment. Wong:Cleave Biosciences: Employment. Sekeres:TetraLogic: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Carew:Boehringer Ingelheim: Research Funding.

scholarly journals Unfolded Protein Response and Crohn’s Diseases: A Molecular Mechanism of Wound Healing in the Gut

2021 ◽  
Author(s):  
Chao Li
Keyword(s):  
Er Stress ◽  
Unfolded Protein Response ◽  
Immune Cell ◽  
Macrophage Polarization ◽  
Intestinal Epithelial ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

Endoplasmic reticulum (ER) stress triggers a series of signaling and transcriptional events termed the unfolded protein response (UPR). Severe ER stress is associated with the development of fibrosis in different organs including lung, liver, kidney, heart, and intestine. ER stress is an essential response of epithelial and immune cells in the pathogenesis of inflammatory bowel disease (IBD) including Crohn&rsquo;s disease. Intestinal epithelial cells are susceptible to ER stress-mediated damage due to secretion of a large amount of proteins that are involved in mucosal defense. In other cells, ER stress is linked to myofibroblast activation, extracellular matrix production, macrophage polarization, and immune cell differentiation. This review focuses on the role of UPR in the pathogenesis in IBD from an immunologic perspective. The roles of macrophage and mesenchymal cells in the UPR from in vitro and in vivo animal models are discussed. The links between ER stress and other signaling pathways such as senescence and autophagy are introduced. Recent advances in the understanding of the epigenetic regulation of UPR signaling are also updated here. The future directions of development of the UPR research and therapeutic strategies to manipulate ER stress levels are also reviewed.

scholarly journals P09.14 Blocking counterregulation of unfolded protein response by targeted protein synthesis inhibition produces highly synergistic cell death in several cancer entities

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A59.1-A59
Author(s):  
F Gsottberger ◽  
C Meier ◽  
S Petkovic ◽  
L Mellenthin ◽  
M Krumbholz ◽  
...  
Keyword(s):  
Cell Death ◽  
Protein Synthesis ◽  
Unfolded Protein Response ◽  
Therapeutic Window ◽  
Protein Synthesis Inhibition ◽  
Synthesis Inhibition ◽  
Unfolded Protein ◽  
Protein Response

BackgroundBecause tumor cells have high proliferation rates the demand for energy on the one hand and proteins on the other hand is high. In line, protein folding machinery of the ER is heavily used. 2-Deoxyglucose (2-DG) not only blocks energy synthesis by inhibiting glycolysis but also blocks synthesis of mannosyl leading to impaired N-linked glycosylation, accumulation of misfolded proteins, and increased unfolded protein response (UPR). However, due to compensatory events, UPR-induced apoptosis is hampered. Therefore, we combined 2-DG with targeted protein synthesis inhibition by immunotoxins, consisting of an antibody and pseudomonas exotoxin, to enhance UPR mediated cell death.Materials and MethodsEstablished cell lines and patient-derived B-ALL samples were treated in vitro with various protein synthesis inhibitors and UPR-inducers. Drug synergy was determined mathematically as fold-increase over additivity. Biochemical studies were performed using western blots. In vivo enhancement was tested using systemic xenograft models.ResultsThe combination of Moxetumomab and 2-DG achieved a two to nine-fold synergy in vitro. Synergy was abrogated by the addition of Mannose suggesting UPR as cause of synergistic cell death. Similarly, Moxetumomab enhanced UPR-inducers Bortezomib and tunicamycin and protein synthesis inhibition by cycloheximide and puromycin enhanced 2-DG suggesting a conserved mechanism. Using HB21, an immunotoxin targeting human transferrin-receptor, breast cancer, hepatocellular carcinoma, and glioblastoma were sensitized to 2-DG induced cell death. Biochemically, 2-DG increased XBP-1-cleavage, expression of pro-apoptotic CHOP and of anti-apoptotic BIP. Moxetumomab, however, blocked the upregulation of BIP while maintaining CHOP correlating with synergistic increase in PARP-cleavage and apoptosis. In two systemic mouse models, bone marrow (BM) lymphoma infiltration was not reduced by 2-DG or tunicamycin alone but was reduced after treatment with Moxetumomab alone by 5-fold in the JeKo-1 and by 16-fold in the Ramos model, respectively. The combination of Moxetumomab and 2-DG achieved a three-fold synergy in the JeKo-1 model and achieved MRD-negative BM status in the Ramos model. Against patient-derived B-ALL of the Burkitt’s type, 2-DG and Moxetumomab were up to 5-fold more active in vitro and up to 7-fold more active in mouse xenografts in vivo.ConclusionsCell death after persisting unfolded protein response is synergistically enhanced by tumor-cell specific inhibition of protein synthesis against four distinct tumor entities at physiologically achievable concentrations. Our approach of immunotoxin-induced targeted protein synthesis inhibition opens a novel, so far undescribed therapeutic window which may warrant clinical evaluation.Disclosure InformationF. Gsottberger: None. C. Meier: None. S. Petkovic: None. L. Mellenthin: None. M. Krumbholz: None. M. Metzler: None. A. Mackensen: None. F. Müller: None.

Irestatin, a potent inhibitor of IRE1α and the unfolded protein response, is a hypoxia-selective cytotoxin and impairs tumor growth

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 3514-3514 ◽  
Author(s):  
D. Feldman ◽  
A. C. Koong
Keyword(s):  
Tumor Growth ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Luciferase Activity ◽  
Single Agent ◽  
Luciferase Reporter ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
Cells Cultured

3514 Background: The unfolded protein response (UPR) is an adaptive response to the toxic accumulation of misfolded proteins in the endoplasmic reticulum (ER), and is activated in solid tumors. IRE1a is a core component of the UPR and responds to ER stress through activation of its dual kinase and endonuclease domains. The IRE1a endonuclease splices the mRNA for XBP-1, generateing a potent transcription factor that is required for tumor growth. Methods: We developed a fibrosarcoma cell line expressing a fusion of unprocessed XBP-1 inserted upstream of firefly luciferase. Under ER stress, IRE1a catalyzes the removal of a 26-nt intronic sequence from the XBP-1 mRNA, introducing a shift in reading frame that permits translation of luciferase. We screened a chemical library of 66,000 small molecules for inhibitors of XBP-luciferase activity and identified 12 molecules, termed irestatins, which consistently inhibited the IRE1a signaling module without affecting the activity of a control CMV-luciferase reporter. We pursued several of the most potent irestatins, including irestatin 9389, which inhibited XBP-luciferase activity with an IC50 of ∼25 nM. Results: Irestatin-mediated inhibition of the IRE1a endonuclease impairs the growth of malignant myeloma cells and inhibits the survival of oxygen-starved tumor cells in vitro. Exposure to irestatin 9389 (2.5 μM) had a negligible effect on the survival of HT1080 cells cultured under normal oxygen conditions. However, in cells cultured under hypoxia for 48 hours, irestatin 9389 inhibited colony formation by nearly 80-fold (48% vs. 0.62%). A two-week course of treatment with single-agent irestatin 9389 (50 mg/kg) administered every other day by i.p. bolus injection was well tolerated and strongly inhibited the growth of subcutaneous HT1080 tumor xenografts (1790 ± 380 mm3 vs. 480 ± 210 mm3; P<0.01). Conclusion: Irestatins define a novel class of hypoxia- and ER stress-selective agents targeted to the underlying physiological response to the tumor microenvironment. Intratumoral inhibition of the UPR can potentiate cell death and impair tumor growth. Molecular intervention against central components of the UPR may represent an effective therapeutic strategy in cancer treatment. No significant financial relationships to disclose.

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