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Article ; Online: A Quantitative Flow Cytometry-Based Method for Autophagy Detection Across the Cell Cycle.

Kaminskyy, Vitaliy O

Methods in molecular biology (Clifton, N.J.)

2021 Volume 2445, Page(s) 65–74

Abstract: Autophagy is deregulated in cancer cells and often activated as a cellular stress response to anticancer therapies. Flow cytometry-based assays enable detection and quantification of various cellular markers in live or fixed cells. Here, a flow cytometry- ...

Abstract	Autophagy is deregulated in cancer cells and often activated as a cellular stress response to anticancer therapies. Flow cytometry-based assays enable detection and quantification of various cellular markers in live or fixed cells. Here, a flow cytometry-based assay to characterize autophagy across the cell cycle is described. This method is based on selective plasma membrane permeabilization with digitonin and extraction of membrane-unbound LC3 protein followed by staining of the autophagosome-bound LC3 protein with antibody and labeling of DNA with propidium iodide. Staining with the LC3 antibody described here can be also combined with the staining of other cellular markers, allowing to quantitatively assess autophagy in relation to different cellular processes by flow cytometry.
MeSH term(s)	Autophagosomes/metabolism ; Autophagy/physiology ; Cell Cycle ; Flow Cytometry/methods ; Microtubule-Associated Proteins/metabolism
Chemical Substances	Microtubule-Associated Proteins
Language	English
Publishing date	2021-12-22
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-2071-7_5
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Cell death-based treatment of various diseases: a fifty-year journey.

Kaminskyy, Vitaliy O / Zhivotovsky, Boris

Cell death & disease

2018 Volume 9, Issue 2, Page(s) 110

MeSH term(s)	Apoptosis ; Diabetes Mellitus/pathology ; Disease ; Humans ; Immunity, Innate ; Necrosis ; Neoplasms/therapy ; Neutrophils/pathology
Language	English
Publishing date	2018-01-25
Publishing country	England
Document type	Editorial ; Introductory Journal Article
ZDB-ID	2541626-1
ISSN	2041-4889 ; 2041-4889
ISSN (online)	2041-4889
ISSN	2041-4889
DOI	10.1038/s41419-017-0168-3
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Knockout validation of LAMP2A antibodies for immunostaining in human cancer cells.

Zhou, Xun / Shirokova, Vera / Kaminskyy, Vitaliy O / Berenger, Eva / Kochetkova, Elena / Norberg, Erik / Genander, Maria / Vakifahmetoglu-Norberg, Helin

Autophagy

2023 Volume 19, Issue 9, Page(s) 2575–2577

Abstract: LAMP2A is the rate-limiting factor of chaperone-mediated autophagy (CMA), a unique selective protein degradative pathway. To date LAMP2A antibodies are not knockout (KO)-validated in human cells. We have recently generated human isoform-specific LAMP2A ... ...

Abstract	LAMP2A is the rate-limiting factor of chaperone-mediated autophagy (CMA), a unique selective protein degradative pathway. To date LAMP2A antibodies are not knockout (KO)-validated in human cells. We have recently generated human isoform-specific LAMP2A KO cells, and here we assessed the specificity of select commercial LAMP2A antibodies on wild-type and LAMP2A KO human cancer cells. While all tested antibodies were suitable for immunoblotting, the anti-LAMP2A antibody (ab18528) is likely to exhibit an off-target reactivity in immunostaining approaches using human cancer cells, and alternative antibodies, which seem more appropriate, are available.
MeSH term(s)	Humans ; Lysosomal-Associated Membrane Protein 2/genetics ; Lysosomal-Associated Membrane Protein 2/metabolism ; Autophagy ; Neoplasms/genetics ; Neoplasms/metabolism ; Chaperone-Mediated Autophagy ; Antibodies ; Lysosomes/metabolism
Chemical Substances	Lysosomal-Associated Membrane Protein 2 ; Antibodies
Language	English
Publishing date	2023-05-16
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2454135-7
ISSN	1554-8635 ; 1554-8627
ISSN (online)	1554-8635
ISSN	1554-8627
DOI	10.1080/15548627.2023.2213515
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Article: Receptor-Mediated Mitophagy Rescues Cancer Cells under Hypoxic Conditions.

Abdrakhmanov, Alibek / Yapryntseva, Maria A / Kaminskyy, Vitaliy O / Zhivotovsky, Boris / Gogvadze, Vladimir

Cancers

2021 Volume 13, Issue 16

Abstract: Targeting mitochondria with thenoyltrifluoroacetone (TTFA), an inhibitor of Complex II in the respiratory chain, stimulated cisplatin-induced apoptosis in various cell lines in normoxia but not in hypoxia. This can be explained by the elimination of ... ...

Abstract	Targeting mitochondria with thenoyltrifluoroacetone (TTFA), an inhibitor of Complex II in the respiratory chain, stimulated cisplatin-induced apoptosis in various cell lines in normoxia but not in hypoxia. This can be explained by the elimination of mitochondria involved in triggering apoptotic cell death by mitophagy, either Parkin-dependent or receptor-mediated. Treatment with TTFA alone or in combination with cisplatin did not cause accumulation of PINK1, meaning that under hypoxic conditions cells survive through activation of a receptor-mediated pathway. Hypoxia triggers the accumulation of BNIP3 and BNIP3L (also known as NIX), key participants in receptor-mediated mitophagy. Under hypoxic conditions, stimulation of autophagy, as assessed by the accumulation of lipidated form of LC3 (LC3II), was observed. To exclude the contribution of canonical macroautophagy in LC3II accumulation, experiments were performed using U1810 cells lacking ATG13, a key enzyme of macroautophagy. Despite the absence of ATG13, hypoxia-mediated accumulation of LC3II was not affected, underlying the importance of the receptor-mediated pathway. In order to prove the protective role of BNIP3 against cisplatin-induced apoptosis, BNIP3-deficient A549 cells were used. Surprisingly, a BNIP3 knockout did not abolish hypoxia-induced protection; however, in cells lacking BNIP3, a compensatory upregulation of BNIP3L was detected. Thus, in the absence of BNIP3, mitophagy could be maintained by BNIP3L and lead to cell death suppression due to the elimination of proapoptotic mitochondria. When both BNIP3 and BNIP3L were knocked out, the inhibitory effect of hypoxia on apoptosis was diminished, although not abolished completely. Undoubtedly, receptor-mediated mitophagy is likely to be one of the mechanisms responsible for cell death suppression under hypoxic conditions.
Language	English
Publishing date	2021-08-10
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2527080-1
ISSN	2072-6694
ISSN	2072-6694
DOI	10.3390/cancers13164027
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Article ; Online: Necroptosis as a Novel Facet of Mitotic Catastrophe.

Egorshina, Aleksandra Yu / Zamaraev, Alexey V / Kaminskyy, Vitaliy O / Radygina, Tatiana V / Zhivotovsky, Boris / Kopeina, Gelina S

International journal of molecular sciences

2022 Volume 23, Issue 7

Abstract: Mitotic catastrophe is a defensive mechanism that promotes elimination of cells with aberrant mitosis by triggering the cell-death pathways and/or cellular senescence. Nowadays, it is known that apoptosis, autophagic cell death, and necrosis could be ... ...

Abstract	Mitotic catastrophe is a defensive mechanism that promotes elimination of cells with aberrant mitosis by triggering the cell-death pathways and/or cellular senescence. Nowadays, it is known that apoptosis, autophagic cell death, and necrosis could be consequences of mitotic catastrophe. Here, we demonstrate the ability of a DNA-damaging agent, doxorubicin, at 600 nM concentration to stimulate mitotic catastrophe. We observe that the inhibition of caspase activity leads to accumulation of cells with mitotic catastrophe hallmarks in which RIP1-dependent necroptotic cell death is triggered. The suppression of autophagy by a chemical inhibitor or
MeSH term(s)	Apoptosis/physiology ; Cell Death ; Humans ; Mitosis ; Necroptosis ; Necrosis
Language	English
Publishing date	2022-03-29
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2019364-6
ISSN	1422-0067 ; 1422-0067 ; 1661-6596
ISSN (online)	1422-0067
ISSN	1422-0067 ; 1661-6596
DOI	10.3390/ijms23073733
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Article ; Online: Free radicals in cross talk between autophagy and apoptosis.

Kaminskyy, Vitaliy O / Zhivotovsky, Boris

Antioxidants & redox signaling

2014 Volume 21, Issue 1, Page(s) 86–102

Abstract: Significance: Oxidative (reactive oxygen species [ROS]) and nitrosative (reactive nitrogen species [RNS]) stress affects many physiological processes, including survival and death. Although high levels of ROS/RNS mainly causes cell death, low levels of ... ...

Abstract	Significance: Oxidative (reactive oxygen species [ROS]) and nitrosative (reactive nitrogen species [RNS]) stress affects many physiological processes, including survival and death. Although high levels of ROS/RNS mainly causes cell death, low levels of free radicals directly modulate the activities of transcriptional factors, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), p53, and nuclear factor (erythroid-derived) 2-like (Nrf2), and regulate numerous protein kinase cascades that participate in the regulation of the cross talk between autophagy and apoptosis. Recent advances: Low levels of ROS modify Atg4 and high mobility group box 1 (HMGB1) proteins, activate AMP-activated protein kinase (AMPK) and apoptosis signal-regulating kinase/c-Jun N-terminal kinase (JNK) pathways, or transactivate various proteins that could upregulate autophagy, leading to reductions in apoptosis. Transactivation of antioxidant genes blocks apoptosis and serves as a feedback loop to reduce autophagy. Free radicals could also activate protein kinase B (PKB, or Akt), preventing both autophagy and apoptosis. Stimulation of nitric oxide formation causes S-nitrosylation of several kinases, including JNK1 and IκB kinase β, which blocks autophagy and could promote apoptosis. However, S-nitrosylation of some proapoptotic proteins could block apoptosis. Critical issues: Endoplasmic reticulum and mitochondria are the main sources of free radicals, which play an essential role in the regulation of apoptosis and autophagy. Oxidation of cardiolipin promotes cytochrome c release and apoptosis that potentially could be inhibited by autophagic clearance of damaged mitochondria. Elimination of damaged mitochondria reduces ROS accumulation, creating a feedback loop that causes inhibition of autophagy. Low levels of RNS could inhibit fission of mitochondria, which would block their degradation by autophagy and spare cells from apoptosis. Future directions: Understanding of mechanisms that regulate the cross talk between cell fates is essential for discovery of therapeutic tools in the strenuous fight against various disorders, including neurodegeneration and cancer.
MeSH term(s)	Animals ; Apoptosis/genetics ; Apoptosis/physiology ; Autophagy/physiology ; Endoplasmic Reticulum/metabolism ; Free Radicals/metabolism ; Humans ; Mitochondria/metabolism
Chemical Substances	Free Radicals
Language	English
Publishing date	2014-07-01
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	1483836-9
ISSN	1557-7716 ; 1523-0864
ISSN (online)	1557-7716
ISSN	1523-0864
DOI	10.1089/ars.2013.5746
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Zs.A 5488: Show issues

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Jg. 1995 - 2021: Lesesall (2.OG)
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Article: EPHA2 Interacts with DNA-PK

Kaminskyy, Vitaliy O / Hååg, Petra / Novak, Metka / Végvári, Ákos / Arapi, Vasiliki / Lewensohn, Rolf / Viktorsson, Kristina

Cancers

2021 Volume 13, Issue 5

Abstract: Ephrin (EFN)/ Erythropoietin-producing human ... ...

Abstract	Ephrin (EFN)/ Erythropoietin-producing human hepatocellular
Language	English
Publishing date	2021-02-28
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2527080-1
ISSN	2072-6694
ISSN	2072-6694
DOI	10.3390/cancers13051010
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Article: Multiplexed electrokinetic sensor for detection and therapy monitoring of extracellular vesicles from liquid biopsies of non-small-cell lung cancer patients

Cavallaro, Sara / Hååg, Petra / Sahu, Siddharth S. / Berisha, Lorenca / Kaminskyy, Vitaliy O. / Ekman, Simon / Lewensohn, Rolf / Linnros, Jan / Viktorsson, Kristina / Dev, Apurba

Biosensors & bioelectronics. 2021 Dec. 01, v. 193

2021

Abstract: Liquid biopsies based on extracellular vesicles (EVs) represent a promising tool for treatment monitoring of tumors, including non-small-cell lung cancers (NSCLC). In this study, we report on a multiplexed electrokinetic sensor for surface protein ... ...

Abstract	Liquid biopsies based on extracellular vesicles (EVs) represent a promising tool for treatment monitoring of tumors, including non-small-cell lung cancers (NSCLC). In this study, we report on a multiplexed electrokinetic sensor for surface protein profiling of EVs from clinical samples. The method detects the difference in the streaming current generated by EV binding to the surface of a functionalized microcapillary, thereby estimating the expression level of a marker. Using multiple microchannels functionalized with different antibodies in a parallel fluidic connection, we first demonstrate the capacity for simultaneous detection of multiple surface markers in small EVs (sEVs) from NSCLC cells. To investigate the prospects of liquid biopsies based on EVs, we then apply the method to profile sEVs isolated from the pleural effusion (PE) fluids of five NSCLC patients with different genomic alterations (ALK, KRAS or EGFR) and applied treatments (chemotherapy, EGFR- or ALK-tyrosine kinase inhibitors). The vesicles were targeted against CD9, as well as EGFR and PD-L1, two treatment targets in NSCLC. The electrokinetic signals show detection of these markers on sEVs, highlighting distinct interpatient differences, e.g., increased EGFR levels in sEVs from a patient with EGFR mutation as compared to an ALK-fusion one. The sensors also detect differences in PD-L1 expressions. The analysis of sEVs from a patient prior and post ALK-TKI crizotinib treatment reveals significant increases in the expressions of some markers (EGFR and PD-L1). These results hold promise for the application of the method for tumor treatment monitoring based on sEVs from patient liquid biopsies.
Keywords	biosensors ; drug therapy ; genomics ; liquids ; lung neoplasms ; lungs ; mutation ; patients ; surface proteins
Language	English
Dates of publication	2021-1201
Publishing place	Elsevier B.V.
Document type	Article
ZDB-ID	1011023-9
ISSN	1873-4235 ; 0956-5663
ISSN (online)	1873-4235
ISSN	0956-5663
DOI	10.1016/j.bios.2021.113568
Database	NAL-Catalogue (AGRICOLA)

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Zs.A 2275: Show issues

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Article ; Online: Characterizing single extracellular vesicles by droplet barcode sequencing for protein analysis.

Banijamali, Mahsan / Höjer, Pontus / Nagy, Abel / Hååg, Petra / Gomero, Elizabeth Paz / Stiller, Christiane / Kaminskyy, Vitaliy O / Ekman, Simon / Lewensohn, Rolf / Karlström, Amelie Eriksson / Viktorsson, Kristina / Ahmadian, Afshin

Journal of extracellular vesicles

2022 Volume 11, Issue 11, Page(s) e12277

Abstract: Small extracellular vesicles (sEVs) have in recent years evolved as a source of biomarkers for disease diagnosis and therapeutic follow up. sEV samples derived from multicellular organisms exhibit a high heterogeneous repertoire of vesicles which current ...

Abstract	Small extracellular vesicles (sEVs) have in recent years evolved as a source of biomarkers for disease diagnosis and therapeutic follow up. sEV samples derived from multicellular organisms exhibit a high heterogeneous repertoire of vesicles which current methods based on ensemble measurements cannot capture. In this work we present droplet barcode sequencing for protein analysis (DBS-Pro) to profile surface proteins on individual sEVs, facilitating identification of sEV-subtypes within and between samples. The method allows for analysis of multiple proteins through use of DNA barcoded affinity reagents and sequencing as readout. High throughput single vesicle profiling is enabled through compartmentalization of individual sEVs in emulsion droplets followed by droplet barcoding through PCR. In this proof-of-concept study we demonstrate that DBS-Pro allows for analysis of single sEVs, with a mixing rate below 2%. A total of over 120,000 individual sEVs obtained from a NSCLC cell line and from malignant pleural effusion (MPE) fluid of NSCLC patients have been analyzed based on their surface proteins. We also show that the method enables single vesicle surface protein profiling and by extension characterization of sEV-subtypes, which is essential to identify the cellular origin of vesicles in heterogenous samples.
MeSH term(s)	Humans ; Extracellular Vesicles/genetics ; Biomarkers/metabolism ; Cell Line ; Membrane Proteins/metabolism
Chemical Substances	Biomarkers ; Membrane Proteins
Language	English
Publishing date	2022-11-03
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2683797-3
ISSN	2001-3078 ; 2001-3078
ISSN (online)	2001-3078
ISSN	2001-3078
DOI	10.1002/jev2.12277
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Article: Detection of Tumor-Associated Membrane Receptors on Extracellular Vesicles from Non-Small Cell Lung Cancer Patients via Immuno-PCR.

Stiller, Christiane / Viktorsson, Kristina / Paz Gomero, Elizabeth / Hååg, Petra / Arapi, Vasiliki / Kaminskyy, Vitaliy O / Kamali, Caroline / De Petris, Luigi / Ekman, Simon / Lewensohn, Rolf / Karlström, Amelie Eriksson

Cancers

2021 Volume 13, Issue 4

Abstract: Precision cancer medicine for non-small-cell lung cancer (NSCLC) has increased patient survival. Nevertheless, targeted agents towards tumor-associated membrane receptors only result in partial remission for a limited time, calling for approaches which ... ...

Abstract	Precision cancer medicine for non-small-cell lung cancer (NSCLC) has increased patient survival. Nevertheless, targeted agents towards tumor-associated membrane receptors only result in partial remission for a limited time, calling for approaches which allow longitudinal treatment monitoring. Rebiopsy of tumors in the lung is challenging, and metastatic lesions may have heterogeneous signaling. One way ahead is to use liquid biopsies such as circulating tumor DNA or small extracellular vesicles (sEVs) secreted by the tumor into blood or other body fluids. Herein, an immuno-PCR-based detection of the tumor-associated membrane receptors EGFR, HER2, and IGF-1R on CD9-positive sEVs from NSCLC cells and pleural effusion fluid (PE) of NSCLC patients is developed utilizing DNA conjugates of antibody mimetics and affibodies, as detection agents. Results on sEVs purified from culture media of NSCLC cells treated with anti-EGFR siRNA, showed that the reduction of EGFR expression can be detected via immuno-PCR. Protein profiling of sEVs from NSCLC patient PE samples revealed the capacity to monitor EGFR, HER2, and IGF-1R with the immuno-PCR method. We detected a significantly higher EGFR level in sEVs derived from a PE sample of a patient with an
Language	English
Publishing date	2021-02-22
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2527080-1
ISSN	2072-6694
ISSN	2072-6694
DOI	10.3390/cancers13040922
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