scholarly journals Complementary Cell-Based High-Throughput Screens Identify Novel Modulators of the Unfolded Protein Response

2011 ◽  
Vol 16 (8) ◽  
pp. 825-835 ◽  
Author(s):  
Andrew M. Fribley ◽  
Patricia G. Cruz ◽  
Justin R. Miller ◽  
Michael U. Callaghan ◽  
Peter Cai ◽  
...  
Keyword(s):  
Unfolded Protein Response ◽  
High Throughput ◽  
High Throughput Screening ◽  
Luciferase Reporter ◽  
Tumor Cell Death ◽  
Unfolded Protein ◽  
Induced Apoptosis ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
The University

Despite advances toward understanding the prevention and treatment of many cancers, patients who suffer from oral squamous cell carcinoma (OSCC) confront a survival rate that has remained unimproved for more than 2 decades, indicating our ability to treat them pharmacologically has reached a plateau. In an ongoing effort to improve the clinical outlook for this disease, we previously reported that an essential component of the mechanism by which the proteasome inhibitor bortezomib (PS-341, Velcade) induced apoptosis in OSCC required the activation of a terminal unfolded protein response (UPR). Predicated on these studies, the authors hypothesized that high-throughput screening (HTS) of large diverse chemical libraries might identify more potent or selective small-molecule activators of the apoptotic arm of the UPR to control or kill OSCC. They have developed complementary cell-based assays using stably transfected CHO-K1 cell lines that individually assess the PERK/eIF2α/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR subpathways. An ˜66 K compound collection was screened at the University of Michigan Center for Chemical Genomics that included a unique library of prefractionated natural product extracts. The mycotoxin methoxycitrinin was isolated from a natural extract and found to selectively activate the CHOP-luciferase reporter at 80 µM. A series of citrinin derivatives was isolated from these extracts, including a unique congener that has not been previously described. In an effort to identify more potent compounds, the authors examined the ability of citrinin and the structurally related mycotoxins ochratoxin A and patulin to activate the UPR. Strikingly, it was found that patulin at 2.5 to 10 µM induced a terminal UPR in a panel of OSCC cells that was characterized by an increase in CHOP, GADD34, and ATF3 gene expression and XBP1 splicing. A luminescent caspase assay and the induction of several BH3-only genes indicated that patulin could induce apoptosis in OSCC cells. These data support the use of this complementary HTS strategy to identify novel modulators of UPR signaling and tumor cell death.

scholarly journals The unfolded protein response controls ER stress-induced apoptosis of lung epithelial cells through angiotensin generation

2016 ◽  
Vol 311 (5) ◽  
pp. L846-L854 ◽  
Author(s):  
Hang Nguyen ◽  
Bruce D. Uhal
Keyword(s):  
Epithelial Cells ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Cathepsin D ◽  
Lung Epithelial Cells ◽  
Chemical Chaperone ◽  
Unfolded Protein ◽  
Induced Apoptosis ◽  
The Unfolded Protein Response ◽  
Protein Response

Recent work from this laboratory showed that endoplasmic reticulum (ER) stress-induced apoptosis of alveolar epithelial cells (AECs) is regulated by the autocrine angiotensin (ANG)II/ANG1-7 system. The proteasome inhibitor MG132 or surfactant protein C (SP-C) BRICHOS domain mutation G100S induced apoptosis in human AECs by activating the proapoptotic cathepsin D and reducing antiapoptotic angiotensin converting enzyme-2 (ACE-2). This study tested the hypothesis that ER stress-induced apoptosis of human AECs might be mediated by influence of the unfolded protein response (UPR) on the autocrine ANGII/ANG1-7 system. A549 cells were challenged with MG132 or SP-C BRICHOS domain mutant G100S to induce ER stress and activation of UPR pathways. The results showed that either MG132 or G100S SP-C mutation activated all three canonical pathways of the UPR (IRE1/XBP1, ATF6, and PERK/eIF2α), which led to a significant increase in cathepsin D or in TACE (an ACE-2 ectodomain shedding enzyme) and eventually caused AEC apoptosis. However, ER stress-induced AEC apoptosis could be prevented by chemical chaperone or by UPR blockers. It is also suggested that ATF6 and IRE1 pathways might play important role in regulation of angiotensin system. These data demonstrate that ER stress induces apoptosis in human AECs through mediation of UPR pathways, which in turn regulate the autocrine ANGII/ANG1-7 system. They also demonstrated that ER stress-induced AEC apoptosis can be blocked by inhibition of UPR signaling pathways.

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.

scholarly journals Mutations of the ELA2 gene found in patients with severe congenital neutropenia induce the unfolded protein response and cellular apoptosis

Blood ◽  
2007 ◽  
Vol 110 (13) ◽  
pp. 4179-4187 ◽  
Author(s):  
David S. Grenda ◽  
Mark Murakami ◽  
Jhuma Ghatak ◽  
Jun Xia ◽  
Laurence A. Boxer ◽  
...  
Keyword(s):  
Unfolded Protein Response ◽  
Strong Support ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia ◽  
Gene Encoding ◽  
Cyclic Neutropenia ◽  
Unfolded Protein ◽  
Induced Apoptosis ◽  
The Unfolded Protein Response ◽  
Protein Response

Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis. Mutations of the ELA2 gene encoding neutrophil elastase (NE) are responsible for most cases of SCN and cyclic neutropenia (CN), a related but milder disorder of granulopoiesis. However, the mechanisms by which these mutations disrupt granulopoiesis are unclear. We hypothesize that the ELA2 mutations result in the production of misfolded NE protein, activation of the unfolded protein response (UPR), and ultimately apoptosis of granulocytic precursors. Expression of mutant NE but not wild-type NE strongly induced BiP/GRP78 mRNA expression and XBP1 mRNA splicing, 2 classic markers of the UPR. The magnitude of UPR activation by a specific ELA2 mutation correlated with its associated clinical phenotype. Consistent with the UPR model, expression of mutant NE in primary human granulocytic precursors increased expression of CHOP (DDITS) and induced apoptosis in a protease-independent fashion. Most strikingly, UPR activation and decreased NE protein expression were detected in primary granulocytic precursors from SCN patients. Collectively, these data provide strong support for a UPR model of SCN disease pathogenesis and place SCN in a growing list of human diseases caused by misfolded proteins.

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