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Mykola Zdioruk

Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)

Recent publications
1.  Zdioruk M., Want A., Mietelska-Porowska A., Laskowska-Kaszub K., Wojsiat J., Klejman A., Użarowska E., Koza P., Olejniczak S., Pikul S., Konopka W., Golab J., Wojda U., A new inhibitor of tubulin polymerization kills multiple cancer cell types and reveals p21-mediated mechanism determining cell death after mitotic catastrophe, Cancers, ISSN: 2072-6694, DOI: 10.3390/cancers12082161, Vol.12, No.8, pp.2161-1-21, 2020

Abstract:
Induction of mitotic catastrophe through the disruption of microtubules is an established target in cancer therapy. However, the molecular mechanisms determining the mitotic catastrophe and the following apoptotic or non-apoptotic cell death remain poorly understood. Moreover, many existing drugs targeting tubulin, such as vincristine, have reduced efficacy, resulting from poor solubility in physiological conditions. Here, we introduce a novel small molecule 2-aminoimidazoline derivative-OAT-449, a synthetic water-soluble tubulin inhibitor. OAT-449 in a concentration range from 6 to 30 nM causes cell death of eight different cancer cell lines in vitro, and significantly inhibits tumor development in such xenograft models as HT-29 (colorectal adenocarcinoma) and SK-N-MC (neuroepithelioma) in vivo. Mechanistic studies showed that OAT-449, like vincristine, inhibited tubulin polymerization and induced profound multi-nucleation and mitotic catastrophe in cancer cells. HeLa and HT-29 cells within 24 h of treatment arrested in G2/M cell cycle phase, presenting mitotic catastrophe features, and 24 h later died by non-apoptotic cell death. In HT-29 cells, both agents altered phosphorylation status of Cdk1 and of spindle assembly checkpoint proteins NuMa and Aurora B, while G2/M arrest and apoptosis blocking was consistent with p53-independent accumulation in the nucleus and largely in the cytoplasm of p21/waf1/cip1, a key determinant of cell fate programs. This is the first common mechanism for the two microtubule-dissociating agents, vincristine and OAT-449, determining the cell death pathway following mitotic catastrophe demonstrated in HT-29 cells.

Keywords:
cancer, chemotherapeutic, microtubule-poison, vincristine, mitotic catastrophe, non-apoptotic cell death, p21, p53

Affiliations:
Zdioruk M. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Want A. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Mietelska-Porowska A. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Laskowska-Kaszub K. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Wojsiat J. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Klejman A. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Użarowska E. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Koza P. - other affiliation
Olejniczak S. - OncoArendi Therapeutics (PL)
Pikul S. - OncoArendi Therapeutics (PL)
Konopka W. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Golab J. - Medical University of Warsaw (PL)
Wojda U. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
2.  Czerkies M., Borzęcka K., Zdioruk M.I., Płóciennikowska A., Sobota A., Kwiatkowska K., An interplay between scavenger receptor A and CD14 during activation of J774 cells by high concentrations of LPS, IMMUNOBIOLOGY, ISSN: 0171-2985, DOI: 10.1016/j.imbio.2013.04.005, Vol.218, pp.1217-1226, 2013

Abstract:
Lipopolysaccharide (LPS) activates macrophages by binding to the TLR4/MD-2 complex and triggers two pro-inflammatory signaling pathways: one relies on MyD88 at the plasma membrane, and the other one depends on TRIF in endosomes. When present in high doses, LPS is internalized and undergoes detoxification. We found that the uptake of a high concentration of LPS (1000 ng/ml) in macrophage-like J774 cells was upregulated upon inhibition of clathrin- and dynamin-mediated endocytosis which, on the other hand, strongly reduced the production of pro-inflammatory mediators TNF-α and RANTES. The binding and internalization of high amounts of LPS was mediated by scavenger receptor A (SR-A) with participation of CD14 without an engagement of TLR4. Occupation of SR-A by dextran sulfate or anti-SR-A antibodies enhanced LPS-induced production of TNF-α and RANTES by about 70%, with CD14 as a limiting factor. Dextran sulfate also elevated the cell surface levels of TLR4 and CD14, which could have contributed to the upregulation of the pro-inflammatory responses. Silencing of SR-A expression inhibited the LPS-triggered TNF-α production whereas RANTES release was unchanged. These data indicate that SR-A is required for maximal production of TNF-α in cells stimulated with LPS, possibly by modulating the cell surface levels of TLR4 and CD14.

Keywords:
CD14, CTX-FITC, Endocytosis, HEPES-buffered saline, LBP, LPS conjugated with Alexa Fluor 488, LPS-AF488, LPS-binding protein, Lipopolysaccharide, PD buffer, SR-A, Scavenger receptor, TLR, poly(I:C), polyinosinic–polycytidylic acid, scavenger receptor A, subunit B of cholera toxin conjugated with FITC, transferrin conjugated with Alexa Fluor 647, transferrin-AF647

Affiliations:
Czerkies M. - other affiliation
Borzęcka K. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Zdioruk M.I. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Płóciennikowska A. - other affiliation
Sobota A. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Kwiatkowska K. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
3.  Kleveta G., Borzęcka K., Zdioruk M., Czerkies M., Kuberczyk H., Sybirna N., Sobota A., Kwiatkowska K., LPS induces phosphorylation of actin-regulatory proteins leading to actin reassembly and macrophage motility, JOURNAL OF CELLULAR BIOCHEMISTRY, ISSN: 0730-2312, DOI: 10.1002/jcb.23330, Vol.113, No.1, pp.80-92, 2012

Abstract:
Upon bacterial infection lipopolysaccharide (LPS) induces migration of monocytes/macrophages to the invaded region and production of pro-inflammatory mediators. We examined mechanisms of LPS-stimulated motility and found that LPS at 100 ng/ml induced rapid elongation and ruffling of macrophage-like J774 cells. A wound-healing assay revealed that LPS also activated directed cell movement that was followed by TNF-α production. The CD14 and TLR4 receptors of LPS translocated to the leading lamella of polarized cells, where they transiently colocalized triggering local accumulation of actin filaments and phosphatidylinositol 4,5-bisphosphate. Fractionation of Triton X-100 cell lysates revealed that LPS induced polymerization of cytoskeletal actin filaments by 50%, which coincided with the peak of cell motility. This microfilament population appeared at the expense of short filaments composing the plasma membrane skeleton of unstimulated cells and actin monomers consisting prior to the LPS stimulation about 60% of cellular actin. Simultaneously with actin polymerization, LPS stimulated phosphorylation of two actin-regulatory proteins, paxillin on tyrosine 118 by 80% and N-WASP on serine 484/485 by 20%, and these events preceded activation of NF-κB. LPS-induced protein phosphorylation and reorganization of the actin cytoskeleton were inhibited by PP2, a drug affecting activity of tyrosine kinases of the Src family. The data indicate that paxillin and N-WASP are involved in the reorganization of actin cytoskeleton driving motility of LPS-stimulated cells. Disturbances of actin organization induced by cytochalasin D did not inhibit TNF-α production suggesting that LPS-induced cell motility is not required for TNF-α release.

Keywords:
ACTIN CYTOSKELETON, CELL MOTILITY, LPS, N-WASP, PAXILLIN, SRC KINASES

Affiliations:
Kleveta G. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Borzęcka K. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Zdioruk M. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Czerkies M. - other affiliation
Kuberczyk H. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Sybirna N. - Ivan Franko Lviv National University (UA)
Sobota A. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Kwiatkowska K. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)

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