Sensitivity of Acute Myelocytic Leukemia Cells to the Dienone Compound VLX1570 Is Associated with Inhibition of the Ubiquitin-Proteasome System

Dienone compounds with a 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore have been widely reported to show tumor cell selectivity. These compounds target the ubiquitin-proteasome system (UPS), known to be essential for the viability of tumor cells. The induction of oxidative stress, depletion of glutathione, and induction of high-molecular-weight (HMW) complexes have also been reported. We here examined the response of acute myeloid leukemia (AML) cells to the dienone compound VLX1570. AML cells have relatively high protein turnover rates and have also been reported to be sensitive to depletion of reduced glutathione. We found AML cells of diverse cytogenetic backgrounds to be sensitive to VLX1570, with drug exposure resulting in an accumulation of ubiquitin complexes, induction of ER stress, and the loss of cell viability in a dose-dependent manner. Caspase activation was observed but was not required for the loss of cell viability. Glutathione depletion was also observed but did not correlate to VLX1570 sensitivity. Formation of HMW complexes occurred at higher concentrations of VLX1570 than those required for the loss of cell viability and was not enhanced by glutathione depletion. To study the effect of VLX1570 we developed a zebrafish PDX model of AML and confirmed antigrowth activity in vivo. Our results show that VLX1570 induces UPS inhibition in AML cells and encourage further work in developing compounds useful for cancer therapeutics.

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β-TrCP-mediated ubiquitination and degradation of Dlg5 regulates hepatocellular carcinoma cell proliferation

Cancer Cell International ◽

10.1186/s12935-019-1029-1 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 1

Author(s):

Dongping Wang ◽

Qi Zhang ◽

Fenfen Li ◽

Chan Wang ◽

Changming Yang ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Xenograft Model ◽

Ubiquitin Proteasome System ◽

Tumor Xenograft ◽

Dependent Manner ◽

Ubiquitin Proteasome ◽

Novel Strategy ◽

Hcc Cells

Abstract Background Discs large homolog 5 (Dlg5) is a member of the membrane-associated guanylate kinase (MAGUK) adaptor family of proteins and its deregulation has been implicated in the malignancy of several cancer types. Dlg5 was down-regulated in hepatocellular carcinoma (HCC) and lower Dlg5 expression was associated with poor survival of HCC patients. However, how to regulate Dlg5 remains largely unknown. Methods The co-immunoprecipitation assay was used to determine the interaction between Dlg5 and β-TrCP. The in vivo ubiquitination assay was performed to determine the regulation of Dlg5 by β-TrCP. CCK-8 and colony formation assay were implemented to detect the biological effect of Dlg5 on the growth of HCC cells in vitro. The effect of Dlg5 on HCC tumor growth in vivo was studied in a tumor xenograft model in mice. Results Here we report that Dlg5 is regulated by the ubiquitin proteasome system and depletion of either Cullin 1 or β-TrCP led to increased levels of Dlg5. β-TrCP regulated Dlg5 protein stability by targeting it for ubiquitination and subsequent destruction in a phosphorylation-dependent manner. We further demonstrated a crucial role of Ser730 in the non-canonical phosphodegron of Dlg5 in governing β-TrCP-mediated Dlg5 degradation. Importantly, failure to degrade Dlg5 significantly inhibited HCC cells proliferation both in vitro and in vivo. Conclusion Collectively, our finding provides a novel molecular mechanism for the negative regulation of Dlg5 by β-TRCP in HCC cells. It further suggests that preventing Dlg5 degradation could be a possible novel strategy for clinical treatment of HCC.

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Kinesin superfamily protein Kif26b links Wnt5a-Ror signaling to the control of cell and tissue behaviors in vertebrates

eLife ◽

10.7554/elife.26509 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 10

Author(s):

Michael W Susman ◽

Edith P Karuna ◽

Ryan C Kunz ◽

Taranjit S Gujral ◽

Andrea V Cantú ◽

...

Keyword(s):

Molecular Mechanisms ◽

Primordial Germ Cells ◽

Ubiquitin Proteasome System ◽

Dependent Manner ◽

Downstream Target ◽

Proteomic Screen ◽

Ubiquitin Proteasome ◽

Canonical Wnt ◽

Kinesin Superfamily

Wnt5a-Ror signaling constitutes a developmental pathway crucial for embryonic tissue morphogenesis, reproduction and adult tissue regeneration, yet the molecular mechanisms by which the Wnt5a-Ror pathway mediates these processes are largely unknown. Using a proteomic screen, we identify the kinesin superfamily protein Kif26b as a downstream target of the Wnt5a-Ror pathway. Wnt5a-Ror, through a process independent of the canonical Wnt/β-catenin-dependent pathway, regulates the cellular stability of Kif26b by inducing its degradation via the ubiquitin-proteasome system. Through this mechanism, Kif26b modulates the migratory behavior of cultured mesenchymal cells in a Wnt5a-dependent manner. Genetic perturbation of Kif26b function in vivo caused embryonic axis malformations and depletion of primordial germ cells in the developing gonad, two phenotypes characteristic of disrupted Wnt5a-Ror signaling. These findings indicate that Kif26b links Wnt5a-Ror signaling to the control of morphogenetic cell and tissue behaviors in vertebrates and reveal a new role for regulated proteolysis in noncanonical Wnt5a-Ror signal transduction.

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Breakdown of Filamentous Myofibrils by the UPS–Step by Step

Biomolecules ◽

10.3390/biom11010110 ◽

2021 ◽

Vol 11 (1) ◽

pp. 110

Author(s):

Dina Aweida ◽

Shenhav Cohen

Keyword(s):

Protein Quality ◽

Protein Structures ◽

Ubiquitin Proteasome System ◽

Surface Proteins ◽

Catalytic Core ◽

Complex Protein ◽

Ubiquitin Proteasome ◽

Aaa Atpases

Protein degradation maintains cellular integrity by regulating virtually all biological processes, whereas impaired proteolysis perturbs protein quality control, and often leads to human disease. Two major proteolytic systems are responsible for protein breakdown in all cells: autophagy, which facilitates the loss of organelles, protein aggregates, and cell surface proteins; and the ubiquitin-proteasome system (UPS), which promotes degradation of mainly soluble proteins. Recent findings indicate that more complex protein structures, such as filamentous assemblies, which are not accessible to the catalytic core of the proteasome in vitro, can be efficiently degraded by this proteolytic machinery in systemic catabolic states in vivo. Mechanisms that loosen the filamentous structure seem to be activated first, hence increasing the accessibility of protein constituents to the UPS. In this review, we will discuss the mechanisms underlying the disassembly and loss of the intricate insoluble filamentous myofibrils, which are responsible for muscle contraction, and whose degradation by the UPS causes weakness and disability in aging and disease. Several lines of evidence indicate that myofibril breakdown occurs in a strictly ordered and controlled manner, and the function of AAA-ATPases is crucial for their disassembly and loss.

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Pyrrolizidine Alkaloids Induce Cell Death in Human HepaRG Cells in a Structure-Dependent Manner

International Journal of Molecular Sciences ◽

10.3390/ijms22010202 ◽

2020 ◽

Vol 22 (1) ◽

pp. 202

Author(s):

Josephin Glück ◽

Julia Waizenegger ◽

Albert Braeuning ◽

Stefanie Hessel-Pras

Keyword(s):

Cell Death ◽

Cell Viability ◽

Pyrrolizidine Alkaloids ◽

Defense Mechanism ◽

Hepatoma Cell ◽

Hepatoma Cell Line ◽

Dependent Manner ◽

Human Hepatoma Cell

Pyrrolizidine alkaloids (PAs) are a group of secondary metabolites produced in various plant species as a defense mechanism against herbivores. PAs consist of a necine base, which is esterified with one or two necine acids. Humans are exposed to PAs by consumption of contaminated food. PA intoxication in humans causes acute and chronic hepatotoxicity. It is considered that enzymatic PA toxification in hepatocytes is structure-dependent. In this study, we aimed to elucidate the induction of PA-induced cell death associated with apoptosis activation. Therefore, 22 structurally different PAs were analyzed concerning the disturbance of cell viability in the metabolically competent human hepatoma cell line HepaRG. The chosen PAs represent the main necine base structures and the different esterification types. Open-chained and cyclic heliotridine- and retronecine-type diesters induced strong cytotoxic effects, while treatment of HepaRG with monoesters did not affect cell viability. For more detailed investigation of apoptosis induction, comprising caspase activation and gene expression analysis, 14 PA representatives were selected. The proapoptotic effects were in line with the potency observed in cell viability studies. In vitro data point towards a strong structure–activity relationship whose effectiveness needs to be investigated in vivo and can then be the basis for a structure-associated risk assessment.

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Identification of the MuRF1 skeletal muscle ubiquitylome through quantitative proteomics

Function ◽

10.1093/function/zqab029 ◽

2021 ◽

Author(s):

Leslie M Baehr ◽

David C Hughes ◽

Sarah A Lynch ◽

Delphi Van Haver ◽

Teresa Mendes Maia ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Atrophy ◽

Potential Role ◽

Ubiquitin Proteasome System ◽

In Vivo Electroporation ◽

Adult Mice ◽

Expression Of Genes ◽

Regulation Of Metabolism ◽

Ubiquitin Proteasome

Abstract MuRF1 (TRIM63) is a muscle-specific E3 ubiquitin ligase and component of the ubiquitin proteasome system. MuRF1 is transcriptionally upregulated under conditions that cause muscle loss, in both rodents and humans, and is a recognized marker of muscle atrophy. In this study, we used in vivo electroporation to determine if MuRF1 overexpression alone can cause muscle atrophy and, in combination with ubiquitin proteomics, identify the endogenous MuRF1 substrates in skeletal muscle. Overexpression of MuRF1 in adult mice increases ubiquitination of myofibrillar and sarcoplasmic proteins, increases expression of genes associated with neuromuscular junction instability, and causes muscle atrophy. A total of 169 ubiquitination sites on 56 proteins were found to be regulated by MuRF1. MuRF1-mediated ubiquitination targeted both thick and thin filament contractile proteins, as well as, glycolytic enzymes, deubiquitinases, p62, and VCP. These data reveal a potential role for MuRF1 in not only the breakdown of the sarcomere, but also the regulation of metabolism and other proteolytic pathways in skeletal muscle.

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Surface-Initiated Atom Transfer Polymerized Anionic Corona on Gold Nanoparticles for Anti-Cancer Therapy

Pharmaceutics ◽

10.3390/pharmaceutics12030261 ◽

2020 ◽

Vol 12 (3) ◽

pp. 261

Author(s):

Wei Mao ◽

Sol Lee ◽

Ji Un Shin ◽

Hyuk Sang Yoo

Keyword(s):

Gold Nanoparticles ◽

Transfection Efficiency ◽

Cancer Therapeutics ◽

Atom Transfer ◽

Layer By Layer ◽

Dependent Manner ◽

Metallic Particles ◽

Anti Cancer

Surface initiated atom transfer radical polymerization (SI-ATRP) documented a simple but efficient technique to grow a dense polymer layer on any surface. Gold nanoparticles (AuNPs) give a broad surface to immobilize sulfhyryl group-containing initiators for SI-ATRP; in addition, AuNPs are the major nanoparticulate carriers for delivery of anti-cancer therapeutics, since they are biocompatible and bioinert. In this work, AuNPs with a disulfide initiator were polymerized with sulfoethyl methacrylate by SI-ATRP to decorate the particles with anionic corona, and branched polyethyeleneimine (PEI) and siRNA were sequentially layered onto the anionic corona of AuNP by electrostatic interaction. The in vitro anti-cancer effect confirmed that AuNP with anionic corona showed higher degrees of apoptosis as well as suppression of the oncogene expression in a siRNA dose-dependent manner. The in vivo study of tumor-bearing nude mice revealed that mice treated with c-Myc siRNA-incorporated AuNPs showed dramatically decreased tumor size in comparison to those with free siRNA for 4 weeks. Furthermore, histological examination and gene expression study revealed that the decorated AuNP significantly suppressed c-Myc expression. Thus, we envision that the layer-by-layer assembly on the anionic brushes can be potentially used to incorporate nucleic acids onto metallic particles with high transfection efficiency.

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Advances in Deubiquitinating Enzyme Inhibition and Applications in Cancer Therapeutics

Cancers ◽

10.3390/cancers12061579 ◽

2020 ◽

Vol 12 (6) ◽

pp. 1579 ◽

Cited By ~ 5

Author(s):

Ainsley Mike Antao ◽

Apoorvi Tyagi ◽

Kye-Seong Kim ◽

Suresh Ramakrishna

Keyword(s):

Protein Interactions ◽

Cancer Therapeutics ◽

Ubiquitin Proteasome System ◽

Deubiquitinating Enzyme ◽

Protein Protein Interactions ◽

Deubiquitinating Enzymes ◽

Cellular Functions ◽

E3 Ligases ◽

Ubiquitin Proteasome ◽

Target Cancer

Since the discovery of the ubiquitin proteasome system (UPS), the roles of ubiquitinating and deubiquitinating enzymes (DUBs) have been widely elucidated. The ubiquitination of proteins regulates many aspects of cellular functions such as protein degradation and localization, and also modifies protein-protein interactions. DUBs cleave the attached ubiquitin moieties from substrates and thereby reverse the process of ubiquitination. The dysregulation of these two paramount pathways has been implicated in numerous diseases, including cancer. Attempts are being made to identify inhibitors of ubiquitin E3 ligases and DUBs that potentially have clinical implications in cancer, making them an important target in the pharmaceutical industry. Therefore, studies in medicine are currently focused on the pharmacological disruption of DUB activity as a rationale to specifically target cancer-causing protein aberrations. Here, we briefly discuss the pathophysiological and physiological roles of DUBs in key cancer-related pathways. We also discuss the clinical applications of promising DUB inhibitors that may contribute to the development of DUBs as key therapeutic targets in the future.

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Wogonoside Inhibits Prostate Cancer Cell Growth and Metastasis via Regulating Wnt/β-Catenin Pathway and Epithelial-Mesenchymal Transition

Pharmacology ◽

10.1159/000502400 ◽

2019 ◽

Vol 104 (5-6) ◽

pp. 312-319 ◽

Cited By ~ 1

Author(s):

Can Wei ◽

Junfeng Jing ◽

Yanbin Zhang ◽

Ling Fang

Keyword(s):

Prostate Cancer ◽

Cell Viability ◽

Epithelial Mesenchymal Transition ◽

Phase Cell ◽

Dependent Manner ◽

Mesenchymal Transition ◽

Pc3 Cells ◽

Gsk 3Β

Background: Wogonoside, an effective component of Scutellaria baicalensis extract, has recently become a hot topic for its newly discovered anticancer efficacy, but the underlying pharmacological mechanism is still unclear. In this study, we tested the inhibitory effects of wogonoside in human prostate cancer PC3 cells in vitro and vivo. Methods: The effects of wogonoside on cell viability, cycle progression, invasion, migration, and apoptosis were assessed in vitro. The levels of proteins in related signaling pathways were detected by western blotting assay. Finally, nude mouse tumorigenicity assay was conducted to detect the anticancer effect of wogonoside in vivo. Results: Wogonoside inhibited cell viability, invasive and migratory ability in a time- and dose-dependent manner. Flow cytometry indicated that wogonoside could induce cell apoptosis and S phase cell-cycle arrest. Mechanically, wogonoside suppressed the Wnt/β-catenin signaling pathway, and the level of p-glycogen synthase kinase-3β (GSK-3β; Ser9) was inhibited by wogonoside. The epithelial-mesenchymal transition (EMT) process was also reversed in PC3 cell line after wogonoside treatment. In vivo experiments showed that wogonoside inhibited tumor growth in xenograft mouse models. Conclusion: These findings revealed that wogonoside could suppress Wnt/β-catenin pathway and reversing the EMT process in PC3 cells. GSK-3β acts as a tumor suppressor in prostate cancer. Wogonoside may serve as an effective agent for treating prostate cancer.

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Degradation of mutant huntingtin via the ubiquitin/proteasome system is modulated by FE65

Biochemical Journal ◽

10.1042/bj20112175 ◽

2012 ◽

Vol 443 (3) ◽

pp. 681-689 ◽

Cited By ~ 15

Author(s):

Wan Ning Vanessa Chow ◽

Hon Wing Luk ◽

Ho Yin Edwin Chan ◽

Kwok-Fai Lau

Keyword(s):

Cell Death ◽

Degradation Mechanism ◽

Neuronal Cell Death ◽

Neuronal Cell ◽

Adaptor Protein ◽

Ubiquitin Proteasome System ◽

Exon 1 ◽

Ubiquitin Proteasome

An unstable expansion of the polyglutamine repeat within exon 1 of the protein Htt (huntingtin) causes HD (Huntington's disease). Mounting evidence shows that accumulation of N-terminal mutant Htt fragments is the source of disruption of normal cellular processes which ultimately leads to neuronal cell death. Understanding the degradation mechanism of mutant Htt and improving its clearance has emerged as a new direction in developing therapeutic approaches to treat HD. In the present study we show that the brain-enriched adaptor protein FE65 is a novel interacting partner of Htt. The binding is mediated through WW–polyproline interaction and is dependent on the length of the polyglutamine tract. Interestingly, a reduction in mutant Htt protein level was observed in FE65-knockdown cells, and the process requires the UPS (ubiquitin/proteasome system). Moreover, the ubiquitination level of mutant Htt was found to be enhanced when FE65 is knocked down. Immunofluroescence staining revealed that FE65 associates with mutant Htt aggregates. Additionally, we demonstrated that overexpression of FE65 increases mutant Htt-induced cell death both in vitro and in vivo. These results suggest that FE65 facilitates the accumulation of mutant Htt in cells by preventing its degradation via the UPS, and thereby enhances the toxicity of mutant Htt.

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Demonstration of the efficacy of compound CFAK-C4 targeting FAK-VEGFR3 protein-protein interaction in gastric cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.e22143 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. e22143-e22143

Author(s):

Elena V. Kurenova ◽

Sartaj Singh Sanghera ◽

Jianqun Liao ◽

Michael Yemma ◽

William G. Cance

Keyword(s):

Gastric Cancer ◽

Tumor Growth ◽

Tumor Progression ◽

Colony Formation ◽

Tumor Regression ◽

Cancer Therapeutics ◽

Scaffolding Protein ◽

Dependent Manner ◽

Gastric Cancer Cells

e22143 Background: While the emerging data strongly suggest that FAK is an excellent target for developmental therapeutics of cancer, kinase inhibitors of FAK have shown crossreactivity with other protein kinases and toxicity in preclinical and clinical studies. It is known that FAK acts pleiotropically, as a kinase and as a scaffolding protein, and our goal is to explore targeting the scaffolding function of FAK to inhibit protein-protein interactions important for tumor progression. Previously, we have shown that FAK physically interacts with VEGFR3 and we identified small molecule inhibitor CFAK-C4 that targets this site of interaction. Both of these kinases are overexpressed in gastric cancers and were found to be independent poor prognostic factors. The prognosis of patients with gastric cancer remains unfavorable and molecular based treatments are necessary for a potential breakthrough in the therapy of this disease. We hypothesize that FAK-VEGFR3 interaction provides essential survival signals for gastric tumor growth and that simultaneous inhibition of these signals will inhibit tumor progression. Methods: Effects of CFAK-C4 on gastric cancer cell lines AGS and NCI-N87 were examined by MTT assay (viability), colony formation assay and Western blotting (phosphorylation, apoptosis). Subcutaneous mouse model was used to demonstrate effect of CFAK-C4 in vivo. Results: CFAK-C4 specifically blocked phosphorylation of VEGFR3 and FAK, directly inhibited cell viability (p<0.05), increased cell detachment and inhibited colony formation in a dose-dependent manner (range 1-100µM). CFAK-C4 (50mg/kg, IP) effectively caused tumor regression in vivo, when administered alone and its effects were synergistic (p<0.05) with chemotherapy. In vivo effects of C4 were confirmed by a decrease in tumor FAK and VEGFR3 phosphorylation, and disruption of their complexes. Conclusions: In this study we have shown that CFAK-C4 inhibits FAK-VEGFR3 signaling in gastric cancer cells and affects tumor growth. This result demonstrates that targeting the scaffolding function of FAK is a unique approach of highly-specific molecular-targeted therapy and can be used to develop oral-based cancer therapeutics.

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