Immunoproteasome Activity and Content Determine Hematopoietic Cell Sensitivity to ONX-0914 and to the Infection of Cells with Lentiviruses

Vagapova, Elmira; Burov, Alexander; Spasskaya, D. S.; Lebedev, Timofey; Astakhova, T. Yu.; Spirin, Pavel; Prassolov, Vladimir; Карпов, В.Л.; Morozov, Alexey

doi:10.3390/cells10051185

Cited by 4 publications

(11 citation statements)

References 60 publications

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“…Cells were homogenized by consecutive freezing/thawing. Chymotrypsyn-like and β5i-specific proteasome activities were measured as described elsewhere ( Vagapova et al, 2021 ) using Suc-LLVY-AMC (Sigma, St. Louis, MO, USA) and Ac-ANW-AMC (Boston Biochem, Cambridge, MA, USA) fluorogenic substrates, correspondingly. Control reactions with 100 nM of the proteasome inhibitor Bortezomib (Selleckchem, Houston, TX, USA) or 1 mM of another proteasome inhibitor MG132 (Santa Cruz Biotechnology Inc., Dallas, Texas, USA) were performed to test nonspecific degradation of substrates.…”

Section: Methodsmentioning

confidence: 99%

“…Human colorectal adenocarcinoma cell line SW480 and embryonic kidney HEK 293T cell line were kindly provided by Dr. Vladimir Prassolov. The SW620B8-mCherry cell line was obtained previously (Burov et al, 2021). The SW480, SW480B8-mCherry and SW620B8-mCherry and HEK 293T cells were maintained in DMEM (Thermo Fisher Scientific, Paisley, Renfrewshire, Scotland, UK).…”

Section: Cell Culturementioning

confidence: 99%

“…The plasmid encoding Cas9D10A, GFP and gRNAs, the donor plasmid used for recombination, as well as the protocol for validation of the obtained cell line were generated previously (Burov et al, 2021). SW480 cells were co-transfected with the plasmids using Mirus TransIT-LT1 reagent (Mirus Bio LLC, Madison, WI, USA) according to manufacturer's instructions.…”

Section: Transfection and Cell Sortingmentioning

confidence: 99%

“…Except use of pro-inflammatory cytokines with pleiotropic effects, currently, no specific drugs that facilitate immunoproteasome synthesis are known. At the same time, several reports indicate altered immunoproteasome subunit expression following treatment of cancer cells with other types of anti-cancer drugs -protein kinase inhibitors that are currently widely used in clinical practice (Burov et al, 2021;Takahashi et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

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Multikinase inhibitors modulate non-constitutive proteasome expression in colorectal cancer cells

Burov,

Grigorieva,

Lebedev

et al. 2024

Front. Mol. Biosci.

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Introduction: Proteasomes are multi-subunit protein complexes responsible for protein degradation in cells. Immunoproteasomes and intermediate proteasomes (together non-constitutive proteasomes) are specific forms of proteasomes frequently associated with immune response, antigen presentation, inflammation and stress. Expression of non-constitutive proteasome subunits has a prognostic value in several types of cancer. Thus, factors that modulate non-constitutive proteasome expression in tumors are of particular interest. Multikinase inhibitors (MKIs) demonstrate promising results in treatment of cancer. At the same time, their immunomodulatory properties and effects on non-constitutive proteasome expression in colorectal cancer cells are poorly investigated.Methods: Proteasome subunit expression in colorectal cancer was evaluated by bioinformatic analysis of available datasets. Two colorectal cancer cell lines, expressing fluorescent non-constitutive proteasomes were treated with multikinase inhibitors: regorafenib and sorafenib. The proteasome subunit expression was assessed by real-time PCR, Western blotting and flow cytometry. The proteasome activity was studied using proteasome activity-based probe and fluorescent substrates. Intracellular proteasome localization was revealed by confocal microscopy. Reactive oxygen species levels following treatment were determined in cells. Combined effect of proteasome inhibition and treatment with MKIs on viability of cells was estimated.Results: Expression of non-constitutive proteasomes is increased in BRAF-mutant colorectal tumors. Regorafenib and sorafenib stimulated the activity and synthesis of non-constitutive proteasomes in examined cell lines. MKIs induced oxidative stress and redistribution of proteasomes within cells. Sorafenib stimulated formation of cytoplasmic aggregates, containing proteolyticaly active non-constitutive proteasomes, while regorafenib had no such effect. MKIs caused no synergistic action when were combined with the proteasome inhibitor.Discussion: Obtained results indicate that MKIs might affect the crosstalk between cancer cells and immune cells via modulation of intracellular proteasome pool. Observed phenomenon should be considered when MKI-based therapy is applied.

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Section: Methodsmentioning

confidence: 99%

Section: Cell Culturementioning

confidence: 99%

Section: Transfection and Cell Sortingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Multikinase inhibitors modulate non-constitutive proteasome expression in colorectal cancer cells

Burov,

Grigorieva,

Lebedev

et al. 2024

Front. Mol. Biosci.

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“…Interestingly, many molecular mechanisms that are used in intracellular stages of virus replication and involved in the response of the human cells to infection are often the same as those which are deregulated in neoplastic cells in different types of cancer. These include cell cycle regulation mechanisms [ 45 ], apoptosis [ 46 , 47 , 48 ], autophagy [ 49 , 50 , 51 ], the DNA repair system [ 52 , 53 , 54 , 55 , 56 , 57 ], cytoskeleton organization mechanisms [ 58 ], the ubiquitin–proteasome system (UPS) [ 59 , 60 , 61 , 62 ], and immune system response [ 63 , 64 ].…”

Section: Introductionmentioning

confidence: 99%

Emetine in Combination with Chloroquine Induces Oncolytic Potential of HIV-1-Based Lentiviral Particles

Spirin

Shyrokova

Vedernikova

et al. 2022

Cells

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Chloroquine and Emetine are drugs used to treat human parasitic infections. In addition, it has been shown that these drugs have an antiviral effect. Both drugs were also found to cause a suppressive effect on the growth of cancer cells of different origins. Here, using the replication-deficient HIV-1-based lentiviral vector particles, we evaluated the ability of the combination of these drugs to reduce viral transduction efficiency. We showed that these drugs act synergistically to decrease cancer cell growth when added in combination with medium containing lentiviral particles. We found that the combination of these drugs with lentiviral particles decreases the viability of treated cells. Taken together, we state the oncolytic potential of the medium containing HIV-1-based particles provoked by the combination of Chloroquine and Emetine.

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Synthesis and Application of a Caged Bioluminescent Probe for the Immunoproteasome

Loy,

Trader

2024

Current Protocols

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Monitoring the catalytic activity of the proteasome and its various isoforms has become increasingly important with the continued development of core particle inhibitors and targeted protein degraders as potential therapies for diseases with high protein accumulation. The immunoproteasome (iCP) is expressed in a variety of diseases due to inflammatory signals, such as interferon‐gamma, that alert the cell to begin generating iCP preferentially over the standard proteasome. There is a need to understand iCP activity and expression both in cells and in vivo because it is becoming a widely targeted isoform in a variety of diseases. Activity‐based probes for the iCP have been developed, but their application has been limited due to their difficult synthesis and choice of fluorescent reporter. There has yet to be a selective iCP probe developed that incorporates a luminescent reporter that could be applied to a variety of different applications. The protocols presented here describe the synthesis of a cleavable activity‐based bioluminescent probe that is selective for the iCP, and the application of the synthesized probe in immunoproteasome activity assays using a luminescent plate reader. Having this bioluminescent reporter, a better understanding of how the iCP is implicated in disease progression, as well as identification of small molecule interactors, can be achieved. © 2024 Wiley Periodicals LLC.Basic Protocol 1: Synthesis of a bioluminescent immunoproteasome probeBasic Protocol 2: Expression of the immunoproteasome in cellsBasic Protocol 3: Immunoproteasome probe application in live cells using a luminescent plate reader

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Immunoproteasome Activity and Content Determine Hematopoietic Cell Sensitivity to ONX-0914 and to the Infection of Cells with Lentiviruses

Cited by 4 publications

References 60 publications

Multikinase inhibitors modulate non-constitutive proteasome expression in colorectal cancer cells

Multikinase inhibitors modulate non-constitutive proteasome expression in colorectal cancer cells

Emetine in Combination with Chloroquine Induces Oncolytic Potential of HIV-1-Based Lentiviral Particles

Synthesis and Application of a Caged Bioluminescent Probe for the Immunoproteasome

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