Publications by authors named "Patrycja Przygodzka"

25 Publications

  • Page 1 of 1

Snail Overexpression Alters the microRNA Content of Extracellular Vesicles Released from HT29 Colorectal Cancer Cells and Activates Pro-Inflammatory State In Vivo.

Cancers (Basel) 2021 Jan 6;13(2). Epub 2021 Jan 6.

Institute of Medical Biology, Polish Academy of Sciences, 93-232 Lodz, Poland.

During metastasis, cancer cells undergo phenotype changes in the epithelial-mesenchymal transition (EMT) process. Extracellular vesicles (EVs) released by cancer cells are the mediators of intercellular communication and play a role in metastatic process. Knowledge of factors that influence the modifications of the pre-metastatic niche for the migrating carcinoma cells is important for prevention of metastasis. We focus here on how cancer progression is affected by EVs released from either epithelial-like HT29-cells or from cells that are in early EMT stage triggered by Snail transcription factor (HT29-Snail). We found that EVs released from HT29-Snail, as compared to HT29-pcDNA cells, have a different microRNA profile. We observed the presence of interstitial pneumonias in the lungs of mice injected with HT29-Snail cells and the percent of mice with lung inflammation was higher after injection of HT29-Snail-EVs. Incorporation of EVs released from HT29-pcDNA, but not released from HT29-Snail, leads to the increased secretion of IL-8 from macrophages. We conclude that Snail modifications of CRC cells towards more invasive phenotype also alter the microRNA cargo of released EVs. The content of cell-released EVs may serve as a biomarker that denotes the stage of CRC and EVs-specific microRNAs may be a target to prevent cancer progression.
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http://dx.doi.org/10.3390/cancers13020172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7830966PMC
January 2021

The New Model of Snail Expression Regulation: The Role of MRTFs in Fast and Slow Endothelial-Mesenchymal Transition.

Int J Mol Sci 2020 Aug 16;21(16). Epub 2020 Aug 16.

Department of Molecular Cell Mechanisms, Medical University of Lodz, 92-215 Lodz, Poland.

Endothelial-mesenchymal transition (EndMT) is a crucial phenomenon in regulating the development of diseases, including cancer metastasis and fibrotic disorders. The primary regulators of disease development are zinc-finger transcription factors belonging to the Snail family. In this study, we characterized the myocardin-related transcription factor (MRTF)-dependent mechanisms of a human promoter regulation in TGF-β-stimulated human endothelial cells. Although in silico analysis revealed that the promoter's regulatory fragment contains one GCCG and two SP1 motifs that could be occupied by MRTFs, the genetic study confirmed that MRTF binds only to SP1 sites to promote snail expression. The more accurate studies revealed that MRTF-A binds to both SP1 elements, whereas MRTF-B to only one (SP1near). Although we found that each MRTF alone is capable of inducing snail expression, the direct cooperation of these proteins is required to reinforce snail expression and promote the late stages of EndMT within 48 hours. Furthermore, genetic and biochemical analysis revealed that MRTF-B alone could induce the late stage of EndMT. However, it requires a prolonged time. Therefore, we concluded that MRTFs might cause EndMT in a fast- and slow-dependent manner. Based on MRTF-dependent Snail upregulation, we recognized that TGF-β1, as an MRTF-B regulator, is involved in slow EndMT induction, whereas TGF-β2, which altered both MRTF-A and MRTF-B expression, promotes a fast EndMT process.
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http://dx.doi.org/10.3390/ijms21165875DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7461591PMC
August 2020

Glypican-1 Level Is Elevated in Extracellular Vesicles Released from MC38 Colon Adenocarcinoma Cells Overexpressing Snail.

Cells 2020 06 30;9(7). Epub 2020 Jun 30.

CNRS UMR 7369, Matrice Extracellulaire et Dynamique Cellulaire (MEDyC), Laboratoire de Biochimie Médicale et Biologie Moléculaire, Université de Reims Champagne Ardenne, 51100 Reims, France.

The transcription factor Snail triggers epithelial-to-mesenchymal transition (EMT), endowing cancer cells with invasive properties during tumor progression. Extracellular vesicles (EVs) released from cancer cells at various stages of cancer progression are known to influence the tumor pre-metastatic niche and metastatic potential. The aim of this study was to analyze the effect of Snail on murine colon adenocarcinoma cells (MC38 line) and on the characteristics of their EVs. Stable clones of Snail-overexpressing MC38 cells were investigated in vitro versus Mock cells. Increased expression of matrix metalloproteinase MMP-14 and augmented activity of MMP-9 and -14 were observed in Snail-MC38 cells. There was no change in the transcriptomic profile of proteoglycans in Snail-MC38 cells; however, the protein level of Glypican-1 (GPC1) was enhanced in EVs released from those cells. Our finding that GPC1 protein level was enhanced in EVs released from MC38 cells that overexpressed Snail and were in an early EMT stage might explain the specificity of the GPC1 biomarker in colon cancer diagnosis. Further, our data suggest that Snail, by changing the level of GPC1 on EVs released by colon cancer cells, may affect the generation of a distant premetastatic niche and metastatic organotropism in colon adenocarcinoma.
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http://dx.doi.org/10.3390/cells9071585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7408449PMC
June 2020

Erratum: Neuromedin U: A Small Peptide in the Big World of Cancer. 2019, , 1312.

Cancers (Basel) 2020 Jan 20;12(1). Epub 2020 Jan 20.

Institute of Medical Biology, Polish Academy of Sciences, 106 Lodowa Str., 93-232 Lodz, Poland.

The authors wish to make the following corrections to this paper [...].
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http://dx.doi.org/10.3390/cancers12010251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7017134PMC
January 2020

Requires Cholesterol Oxidase to Disrupt TLR2 Signalling in Human Macrophages.

Mediators Inflamm 2019 1;2019:2373791. Epub 2019 Dec 1.

Institute of Medical Biology, Polish Academy of Sciences, Lodz, Poland.

This study tested the hypothesis that (Mtb) uses a cholesterol oxidase enzyme (ChoD) to suppress a toll-like receptor type 2- (TLR2-) dependent signalling pathway to modulate macrophages' immune response. We investigated the impact of Mtb possessing or lacking ChoD as well as TBChoD recombinant protein obtained from Mtb on the expression and activation of two key intracellular proteins involved in TLR2 signalling in human macrophages. Finally, the involvement of TLR2-related signalling proteins in an inflammatory/immunosuppressive response of macrophages to Mtb was evaluated. We demonstrate that wild-type Mtb but not the ∆ mutant decreased the cytosolic IRAK4 and TRAF6 protein levels while strongly enhancing and mRNA levels in macrophages. Our data show that the TLR2 present on the surface of macrophages are involved in disturbing the signalling pathway by wild-type Mtb. Moreover, recombinant TBChoD effectively decreased the cytosolic level of TRAF6 and lowered the phosphorylation of IRAK4, which strongly confirm an involvement of cholesterol oxidase in affecting the TLR2-related pathway by Mtb. Wild-type Mtb induced an immunosuppressive response of macrophages in an IRAK4- and TRAF6-dependent manner as measured by interleukin 10 production. In conclusion, ChoD is a virulence factor that enables Mtb to disturb the TLR2-related signalling pathway in macrophages and modulate their response.
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http://dx.doi.org/10.1155/2019/2373791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6913169PMC
June 2020

MS CD49dCD154 Lymphocytes Reprogram Oligodendrocytes into Immune Reactive Cells Affecting CNS Regeneration.

Cells 2019 11 25;8(12). Epub 2019 Nov 25.

Department of Neurology, Laboratory of Neuroimmunology, Medical University of Lodz, Poland, Pomorska Str. 251, 92-213 Lodz, Poland.

The critical aspect in multiple sclerosis (MS) progression involves insufficient regeneration of CNS resulting from deficient myelin synthesis by newly generated oligodendrocytes (OLs). Although many studies have focused on the role of autoreactive lymphocytes in the inflammatory-induced axonal loss, the problem of insufficient remyelination and disease progression is still unsolved. To determine the effect of myelin-specific lymphocytes on OL function in MS patients and in a mouse model of MS, we cultured myelin induced MS CD49dCD154 circulating lymphocytes as well as Experimental Autoimmune Encephalomyelitis (EAE) mouse brain-derived T and memory B cells with maturing oligodendrocyte precursor cells (OPCs). We found that myelin-specific CD49dCD154 lymphocytes affected OPC maturation toward formation of immune reactive OLs. Newly generated OLs were characterized by imbalanced myelin basic protein (MBP) and proteolipid protein (PLP) production as well as proinflammatory chemokine/cytokine synthesis. The analysis of cellular pathways responsible for OL reprogramming revealed that CD49dCD154 lymphocytes affected miRNA synthesis by dysregulation of polymerase II activity. miR-665 and ELL3 turned out to be the main targets of MS myelin-specific lymphocytes. Neutralization of high intracellular miR-665 concentration restored miRNA and MBP/PLP synthesis. Together, these data point to new targets for therapeutic intervention promoting CNS remyelination.
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http://dx.doi.org/10.3390/cells8121508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6953114PMC
November 2019

Neuromedin U: A Small Peptide in the Big World of Cancer.

Cancers (Basel) 2019 Sep 5;11(9). Epub 2019 Sep 5.

Institute of Medical Biology, Polish Academy of Sciences, 106 Lodowa Str, 93-232 Lodz, Poland.

Neuromedin U (NMU), a neuropeptide isolated from porcine spinal cord and named because of its activity as a rat uterus smooth muscle contraction inducer, is emerging as a new player in the tumorigenesis and/or metastasis of many types of cancers. Expressed in a variety of tissues, NMU has been shown to possess many important activities in the central nervous system as well as on the periphery. Along with the main structural and functional features of NMU and its currently known receptors, we summarized a growing number of recently published data from different tissues and cells that associate NMU activity with cancer development and progression. We ask if, based on current reports, NMU can be included as a marker of these processes and/or considered as a therapeutic target.
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http://dx.doi.org/10.3390/cancers11091312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6770777PMC
September 2019

Naturally Occurring Nervonic Acid Ester Improves Myelin Synthesis by Human Oligodendrocytes.

Cells 2019 07 29;8(8). Epub 2019 Jul 29.

Department of Neurology, Laboratory of Neuroimmunology, Medical University of Lodz, Pomorska Str. 251, 92-213 Lodz, Poland.

The dysfunction of oligodendrocytes (OLs) is regarded as one of the major causes of inefficient remyelination in multiple sclerosis, resulting gradually in disease progression. Oligodendrocytes are derived from oligodendrocyte progenitor cells (OPCs), which populate the adult central nervous system, but their physiological capability to myelin synthesis is limited. The low intake of essential lipids for sphingomyelin synthesis in the human diet may account for increased demyelination and the reduced efficiency of the remyelination process. In our study on lipid profiling in an experimental autoimmune encephalomyelitis brain, we revealed that during acute inflammation, nervonic acid synthesis is silenced, which is the effect of shifting the lipid metabolism pathway of common substrates into proinflammatory arachidonic acid production. In the experiments on the human model of maturating oligodendrocyte precursor cells (hOPCs) in vitro, we demonstrated that fish oil mixture (FOM) affected the function of hOPCs, resulting in the improved synthesis of myelin basic protein, myelin oligodendrocyte glycoprotein, and proteolipid protein, as well as sphingomyelin. Additionally, FOM reduces proinflammatory cytokines and chemokines, and enhances fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF) synthesis by hOPCs was also demonstrated. Based on these observations, we propose that the intake of FOM rich in the nervonic acid ester may improve OL function, affecting OPC maturation and limiting inflammation.
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http://dx.doi.org/10.3390/cells8080786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721595PMC
July 2019

Regulation of miRNAs by Snail during epithelial-to-mesenchymal transition in HT29 colon cancer cells.

Sci Rep 2019 02 15;9(1):2165. Epub 2019 Feb 15.

Institute of Medical Biology, PAS, 106 Lodowa Street, 93232, Lodz, Poland.

Epithelial-to-mesenchymal transition (EMT) in cancer cells, represents early stages of metastasis and is a promising target in colorectal cancer (CRC) therapy. There have been many attempts to identify markers and key pathways induced throughout EMT but the process is complex and depends on the cancer type and tumour microenvironment. Here we used the colon cancer cell line HT29, which stably overexpressed Snail, the key transcription factor in early EMT, as a model for colorectal adenocarcinoma cells with a pro-metastatic phenotype. We investigated miRNA expression regulation during that phenotypic switching. We found that overexpression of Snail in HT29 cells triggered significant changes in individual miRNA levels but did not change the global efficiency of miRNA processing. Snail abundance repressed the expression of miR-192 and miR-194 and increased miR-205, let-7i and SNORD13 levels. These identified changes correlated with the reported transcriptomic alterations in Snail-overexpressing HT29 cells. We also investigated how Snail affected the miRNA content of extracellular vesicles (EVs) released from HT29 cells. Our data suggest that the presence of Snail significantly alters the complex mRNA/miRNA interactions in the early steps of metastasis and also has an impact on the content of EVs released from HT29 cells.
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http://dx.doi.org/10.1038/s41598-019-39200-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6377707PMC
February 2019

C5a-Preactivated Neutrophils Are Critical for Autoimmune-Induced Astrocyte Dysregulation in Neuromyelitis Optica Spectrum Disorder.

Front Immunol 2018 23;9:1694. Epub 2018 Jul 23.

Department of Neurology, Laboratory of Neuroimmunology, Medical University of Lodz, Lodz, Poland.

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune neuroinflammatory disease. In contrast to multiple sclerosis, autoantibodies against aquaporin-4 (AQP4) expressed on astrocytic end-feet have been exclusively detected in sera of NMOSD patients. Several lines of evidence suggested that anti-AQP4 autoantibodies are pathogenic, but the mechanism triggering inflammation, impairment of astrocyte function, and the role of neutrophils presented in NMOSD cerebrospinal fluid remains unknown. In this study, we tested how human neutrophils affect astrocytes in the presence of anti-AQP4 Ab-positive serum derived from NMOSD patients. An model of inflammation consisted of human astrocyte line, NMOSD serum, and allogenic peripheral blood neutrophils from healthy individuals. We showed evidence of pathogenicity of NMOSD serum, which by consecutive action of anti-AQP4 Abs, complement system, and neutrophils affected astrocyte function. Anti-AQP4 Ab binding astrocytes initiated two parallel complementary reactions. The first one was dependent on the complement cytotoxicity C5b-9 complex formation, and the second one on the reverse of astrocyte glutamate pump into extracellular space by C5a-preactivated neutrophils. As a consequence, astrocytes were partially destroyed; however, a major population of astrocytes polarized into proinflammatory cells which were characterized by pathological glutamate removal from extracellular space.
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http://dx.doi.org/10.3389/fimmu.2018.01694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6065055PMC
July 2018

HMEC-1 adopt the mixed amoeboid-mesenchymal migration type during EndMT.

Eur J Cell Biol 2017 Jun 24;96(4):289-300. Epub 2017 Apr 24.

Institute of Medical Biology, PAS, 106 Lodowa Street, 93232 Lodz, Poland. Electronic address:

The contribution of endothelial cells to scar and fibrotic tissue formation is undisputedly connected to their ability to undergo the endothelial-to-mesenchymal transition (EndMT) towards fibroblast phenotype-resembling cells. The migration model of fibroblasts and fibroblast-resembling cells is still not fully understood. It may be either a Rho/ROCK-independent, an integrin- and MMP-correlated ECM degradation-dependent, a mesenchymal model or Rho/ROCK-dependent, integrin adhesion- and MMP activity-independent, an amoeboid model. Here, we hypothesized that microvascular endothelial cells (HMEC-1) undergoing EndMT adopt an intermediate state of drifting migration model between the mesenchymal and amoeboid protrusive types in the early stages of fibrosis. We characterized the response of HMEC-1 to TGF-β2, a well-known mediator of EndMT within the microvasculature. We observed that TGF-β2 induces up to an intermediate mesenchymal phenotype in HMEC-1. In parallel, MMP-2 is upregulated and is responsible for most proteolytic activity. Interestingly, the migration of HMEC-1 undergoing EndMT is dependent on both ECM degradation and invadosome formation associated with MMP-2 proteolytic activity and Rho/ROCK cytoskeleton contraction. In conclusion, the transition from mesenchymal towards amoeboid movement highlights a molecular plasticity mechanism in endothelial cell migration in skin fibrosis.
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http://dx.doi.org/10.1016/j.ejcb.2017.04.002DOI Listing
June 2017

Neuromedin U is upregulated by Snail at early stages of EMT in HT29 colon cancer cells.

Biochim Biophys Acta 2016 11 20;1860(11 Pt A):2445-2453. Epub 2016 Jul 20.

Institute of Medical Biology, PAS, 106 Lodowa Street, 93232 Lodz, Poland. Electronic address:

Background: The epithelial-mesenchymal transition (EMT) is considered a core process that facilitates the escape of cancer cells from the primary tumor site. The transcription factor Snail was identified as a key regulator of EMT; however, the cascade of regulatory events leading to metastasis remains unknown and new predictive markers of the process are awaited.

Methods: Gene expressions were analysed using real-time PCR, protein level by Western immunoblotting and confocal imaging. The motility of the cells was examined using time-lapse microscopy. Affymetrix GeneChip Human Genome U133 Plus 2.0 analysis was performed to identify transcriptomic changes upon Snail. Snail silencing was performed using siRNA nucleofection. NMU detection was performed by ELISA.

Results: HT29 cells overexpressing Snail showed changed morphology, functions and transcriptomic profile indicating EMT induction. Changes in expression of 324 genes previously correlated with cell motility were observed. Neuromedin U was the second highest upregulated gene in HT29-Snail cells. This increase was validated by real-time PCR. Additionally elevated NMU protein was detected by ELISA in cell media.

Conclusions: These results show that Snail in HT29 cells regulates early phenotype conversion towards an intermediate epithelial state. We provided the first evidence that neuromedin U is associated with Snail regulatory function of metastatic induction in colon cancer cells.

General Significance: We described the global, early transcriptomic changes induced through Snail in HT29 colon cancer cells and suggested NMU involvement in this process.
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http://dx.doi.org/10.1016/j.bbagen.2016.07.012DOI Listing
November 2016

Lumican Inhibits SNAIL-Induced Melanoma Cell Migration Specifically by Blocking MMP-14 Activity.

PLoS One 2016 1;11(3):e0150226. Epub 2016 Mar 1.

CNRS UMR 7369, Matrice Extracellulaire et Dynamique Cellulaire (MEDyC), Université de Reims Champagne Ardenne, Laboratoire de Biochimie Médicale et de Biologie Moléculaire, Reims, France.

Lumican, a small leucine rich proteoglycan, inhibits MMP-14 activity and melanoma cell migration in vitro and in vivo. Snail triggers epithelial-mesenchymal transitions endowing epithelial cells with migratory and invasive properties during tumor progression. The aim of this work was to investigate lumican effects on MMP-14 activity and migration of Snail overexpressing B16F1 (Snail-B16F1) melanoma cells and HT-29 colon adenocarcinoma cells. Lumican inhibits the Snail induced MMP-14 activity in B16F1 but not in HT-29 cells. In Snail-B16F1 cells, lumican inhibits migration, growth, and melanoma primary tumor development. A lumican-based strategy targeting Snail-induced MMP-14 activity might be useful for melanoma treatment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0150226PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4773148PMC
July 2016

Antitumoral Activity Of Nitric Oxide-Releasing Compounds.

Redox Biol 2015 08 30;5:420. Epub 2015 Dec 30.

Institute of Medical Biology, Polish Academy of Sciences, Lodz, Poland.

Background: Despite significant improvements in the conventional anti-ovarian cancer therapies, tumor cell resistance to various cytostatic drugs remains a relevant problem. Therefore, the new cancer treatment strategies are being developed. Among many agents that have been studied for their potential anti-cancer activity, the most promising are the nitric oxide (NO) donors-syntethic compounds that release NO in vivo and/or in vitro.

Aim: We have evaluated the effect of NO donors on the SK-OV-3 and OVCAR-3 ovarian cancer cell lines. We assessed some of the cancer cells' specific features: the uncontrolled proliferation, over-activation of particular signaling proteins, high resistance to therapeutics and elevated expression and secretion of invasiveness/metastatic factors.

Methods: Two members of NONOates family were used: SPER/NO and DETA/NO. Cancer cell lines were cultured with different concentrations of NO donors. The cytotoxic, pro-apoptotic activity of NO donors and their impact on the phosphorylation status of STAT-3 and AKT in cells were determined. The expression of VEGF-A, MMPs and TGF-β was also evaluated.

Results: NO donors inhibited ovarian cancer cells growth making them also more susceptible to the cisplatin cytotoxic activity. Moreover, both NO donors induced apoptosis of cells and decreased activity of signaling proteins (STAT3 and AKT). Similarly, SPER/NO and DETA/NO lowered the secretion of pro-metastatic factors, responsible for cancer cells invasiveness.

Conclusions: The obtained results show that both NO donors demonstrated a wide range of action on both ovarian cancer cell lines. Therefore, they have a high potential of being a supporting compounds in the cancer therapies.
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http://dx.doi.org/10.1016/j.redox.2015.09.031DOI Listing
August 2015

Inhibition of cyclooxygenase-2 causes a decrease in coronary flow in diabetic mice. The possible role of PGE2 and dysfunctional vasodilation mediated by prostacyclin receptor.

J Physiol Biochem 2015 Sep 5;71(3):351-8. Epub 2015 May 5.

Department of Haemostasis and Haemostatic Disorders, Chair of Biomedical Sciences, Medical University of Lodz, Lodz, 92-215, Poland,

Several lines of evidence suggest that cyclooxygenase-2 (COX-2) activity can have a beneficial role in the maintenance of vascular tone of the blood vessels in diabetes. Specifically, the increased production of prostacyclin (PGI2) and prostaglandin E2 (PGE2), mediated by COX-2, has been suggested to compensate for decreased synthesis of nitric oxide (NO). The study investigates whether inhibition of COX-2 may reduce the coronary flow in diabetic animals and may also lead to decreased synthesis of prostaglandins. Mice aged 18-20 weeks were used for the study: those with leptin receptor deficiency (db/db) served as a model of diabetes while heterozygous (db/+) mice served as controls. Coronary flow was measured by the Langendorff method, and prostaglandin synthesis by myocardia was assayed in heart perfusates. COX-2 inhibition was found to reduce basal coronary flow in db/db mice but had no effect in db/+ mice. Secretion of PGE2 was found to be higher in db/db mice, while prostacyclin synthesis did not differ. COX-2 inhibition decreased production of both prostaglandins to similar levels in both groups. The use of ONO-1301, a specific agonist for the prostacyclin receptor revealed that vasodilating responses mediated by the receptor were impaired in db/db mice. The expression levels of the receptor in cardiac tissue did not differ between the groups. It is concluded that the increased COX-2 contribution to vasodilation in diabetic animals appears to be partially a result of increased COX-2-dependent synthesis of PGE2 and also may be caused by impaired vasodilation mediated by the prostacyclin receptor.
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http://dx.doi.org/10.1007/s13105-015-0415-yDOI Listing
September 2015

Downregulation of striatin leads to hyperphosphorylation of MAP2, induces depolymerization of microtubules and inhibits proliferation of HEK293T cells.

FEBS Lett 2015 Jan 10;589(2):222-30. Epub 2014 Dec 10.

Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, 112 Sienkiewicza St., 90-363 Lodz, Poland. Electronic address:

Microtubules are tubular polymers of α/β-tubulin that are involved in the maintenance of cell shape, motility, and intracellular transport and in the segregation of chromosomes during cell division. Microtubules are dynamic structures, and their assembly is regulated by phosphoproteins called microtubule-associated proteins (MAPs). We propose that striatin, a protein belonging to the striatin family of proteins, is involved in regulation of microtubules. In HEK293T cells, striatin colocalizes with microtubules and stably associates with PP2Ac. Inhibition of striatin expression results in hyperphosphorylation of MAP2 and destabilizes microtubules. Striatin-induced destabilization of microtubules inhibited the proliferation of HEK293T cells and caused the accumulation of cells in the G0/G1 phase of the cell cycle. These results suggest that the PP2A/striatin complex modulates microtubule dynamics by regulating MAP2 phosphorylation.
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http://dx.doi.org/10.1016/j.febslet.2014.12.003DOI Listing
January 2015

Ficolin-3 activity towards the opportunistic pathogen, Hafnia alvei.

Immunobiology 2015 Jan 19;220(1):117-23. Epub 2014 Aug 19.

Institute of Medical Biology, Polish Academy of Sciences, Lodowa 106, 93-232 Lodz, Poland.

Ficolin-3 (also called H-ficolin or Hakata antigen) is a complement-activating pattern recognition molecule, possessing a fibrinogen-like domain involved in carbohydrate binding. Amongst human ficolins, Ficolin-3 has the highest concentration in serum and is the most potent lectin pathway activator in vitro. Evidence for its physiological function is sparse, although its deficiency has been suggested to increase susceptibility to infections. The specificity of Ficolin-3 is poorly characterized and currently few ligands are known. Here we report agglutination of Hafnia alvei, a Gram-negative enteric commensal bacterium and opportunist pathogen, in the presence of recombinant Ficolin-3 and calcium. Ficolin-3 also augmented phagocytosis of H. alvei by macrophages and displayed bactericidal activity. Additionally, Ficolin-3 inhibited host cells' response to TLR4/MD-2/CD14-LPS dependent NF-κB activation. This is the first demonstration of protective activity of Ficolin-3 against a human bacterial pathogen. Although human Ficolin-3 does not recognise and bind to common pathogenic bacteria, it could be an important component of innate immunity providing protection, for example, from commensal flora that can cause extraintestinal, opportunistic infections.
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http://dx.doi.org/10.1016/j.imbio.2014.08.012DOI Listing
January 2015

Nitric oxide donors reduce the invasion ability of ovarian cancer cells in vitro.

Anticancer Drugs 2014 Nov;25(10):1141-51

aInstitute of Medical Biology, Polish Academy of Sciences bDepartment of Gynecology and Gynecologic Oncology, Polish Mother's Memorial Hospital-Research Institute, Lodz, Poland.

The most important factors involved in tumor metastasis and angiogenesis are metalloproteinases (MMPs), vascular endothelial growth factor, and multifunctional transforming growth factor β1. These factors are responsible for extracellular matrix degradation, induction of vascular permeability, and enhancement of tumor cells' invasion and metastasis. Elevated expression and secretion of the above-mentioned factors are correlated with the higher aggressiveness of tumors and low patient survival for example, patients with ovarian cancer. Therefore, regulation of the expression, secretion, and activity of these factors is still considered a potent target for therapeutic intervention in cancer patients. Nitric oxide (NO) donors belong to the class of agents with multivalent targeted activities in cancer cells and are considered potential anticancer therapeutics. Our studies have shown that NO donors such as spermine/NO and diethylenetriamine/NO decrease the secretion of vascular endothelial growth factor-A from the OVCAR-3 ovarian cancer cell line, but not from the SK-OV-3 ovarian cancer cell line. The release of MMP-2 from both cell lines was reduced in a soluble guanylate cyclase-dependent manner by spermine/NO and diethylenetriamine/NO. Nevertheless, MMP-2 activity was only affected in SK-OV-3 cells. Both NO donors reduced the transmigration of the ovarian cancer cell lines. We did not observe any significant effect of spermine/NO and diethylenetriamine/NO on mRNA expression of the tested aggressiveness factors. In conclusion, our data indicated that NO donors reduced the metastatic potential of ovarian cancer cells, but its impact is rather low and requires high concentrations of donors. Moreover, both the tested cell lines differed in the susceptibility to NO donors.
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http://dx.doi.org/10.1097/CAD.0000000000000149DOI Listing
November 2014

NFAT2 regulates COX-2 expression and modulates the integrin repertoire in endothelial cells at the crossroads of angiogenesis and inflammation.

Exp Cell Res 2014 Jun 18;324(2):124-36. Epub 2014 Mar 18.

Institute of Medical Biology, Polish Academy of Science, Lodz 93-232, Poland; Department of Molecular and Medical Biophysics, Medical University of Lodz, Lodz, Poland.

The mechanisms controlling the switch between the pro-angiogenic and pro-inflammatory states of endothelial cells are still poorly understood. In this paper, we show that: (a) COX-2 expression induced by VEGF-A is NFAT2-dependent; and (b) the integrin profile in endothelial cells induced by the pro-angiogenic VEGF-A is distinct from that brought on by the inflammatory cytokine TNF-α. Two groups of integrin subunits specifically upregulated over time by both cytokines were identified using RT-PCR and Western Immunoblotting. The first group included α4, α5, α6, and β5 subunits that were upregulated by VEGF-A; the second group consisted of αV and β3 induced by TNF-α. Both cytokines significantly enhanced the expression of β1 and modulated α2 mRNA. In contrast to TNF-α, VEGF-A induction of integrin subunits depended on the activation of the calcineurin/NFAT pathway. Both calcineurin inhibitors (cyclosporineA and 11R-VIVIT) and downregulation of NFAT2 with specific siRNA decreased induction of integrin subunits. This process of induction could be increased by upregulation of NFAT2 by pBJ5-NFAT2 transfection. This suggests that NFAT2 mediates VEGF-induced upregulation of integrin subunit synthesis by providing a constant supply of newly synthesized "refreshed" mature integrin receptors, particularly α2β1, α5β1, α4β1, α6β1 and αVβ5, which are involved at different stages of angiogenesis.
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http://dx.doi.org/10.1016/j.yexcr.2014.03.008DOI Listing
June 2014

Secretion of SerpinB2 from endothelial cells activated with inflammatory stimuli.

Exp Cell Res 2013 May 7;319(8):1213-9. Epub 2013 Mar 7.

Institute of Medical Biology, Polish Academy of Sciences, Lodz, Poland.

Due to the lack of an N-terminal signal peptide, SerpinB2 (plasminogen activator inhibitor type 2) accumulates in cells and only a small percentage of it is secreted. The extracellular concentration of SerpinB2 significantly increases during inflammation. In the present study we investigated the mechanism with which SerpinB2 can be secreted from endothelial cells activated with LPS. We evaluated the intracellular distribution of SerpinB2 by double immunogold labeling followed by a high resolution electron microscopy analysis. We found that SerpinB2 gathers in the vesicular structures and in the endothelial cell periphery. These vesicles stained positive for the trans-Golgi network marker TGN46, which is consistent with their formation by the endoplasmatic reticulum (ER) and Golgi-dependent pathways. SerpinB2 was delivered to the plasma membrane, apparently together with TGN46 in the same vesicles, which after fusion with the membranes released cargo. Secretion of SerpinB2 was partially inhibited by brefeldin A. The secreted SerpinB2 was predominantly in its nonglycosylated 43kDa form as evaluated by Western immunoblotting. Our data suggest that increased expression of SerpinB2 by an inflammatory stimulus is sufficient to generate structures that resemble secretory vesicles. These vesicles may represent the mechanism by which high local concentrations of SerpinB2 are released at inflammation sites from endothelial cells.
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http://dx.doi.org/10.1016/j.yexcr.2013.02.018DOI Listing
May 2013

Association of plasminogen activator inhibitor type 2 (PAI-2) with proteasome within endothelial cells activated with inflammatory stimuli.

J Biol Chem 2011 Dec 5;286(50):43164-71. Epub 2011 Oct 5.

Institute of Medical Biology, Polish Academy of Sciences, Lodz, Poland.

Quiescent endothelial cells contain low concentrations of plasminogen activator inhibitor type 2 (PAI-2). However, its synthesis can be rapidly stimulated by a variety of inflammatory mediators. In this study, we provide evidence that PAI-2 interacts with proteasome and affects its activity in endothelial cells. To ensure that the PAI-2·proteasome complex is formed in vivo, both proteins were coimmunoprecipitated from endothelial cells and identified with specific antibodies. The specificity of this interaction was evidenced after (a) transfection of HeLa cells with pCMV-PAI-2 and coimmunoprecipitation of both proteins with anti-PAI-2 antibodies and (b) silencing of the PAI-2 gene using specific small interfering RNA (siRNA). Subsequently, cellular distribution of the PAI-2·proteasome complexes was established by immunogold staining and electron microscopy analyses. As judged by confocal microscopy, both proteins appeared in a diffuse cytosolic pattern, but they also could be found in a dense perinuclear and nuclear location. PAI-2 was not polyubiquitinated, suggesting that it bound to proteasome not as the substrate but rather as its inhibitor. Consistently, increased PAI-2 expression (a) abrogated degradation of degron analyzed after cotransfection of HeLa cells with pCMV-PAI-2 and pd2EGFP-N1, (b) prevented degradation of p53, as evidenced both by confocal microscopy and Western immunoblotting, and (c) inhibited proteasome cleavage of specific fluorogenic substrate. This suggests that PAI-2, in endothelial cells induced with inflammatory stimuli, can inhibit proteasome and thus tilt the balance favoring proapoptotic signaling.
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http://dx.doi.org/10.1074/jbc.M111.245647DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3234800PMC
December 2011

Matrin 3 as a key regulator of endothelial cell survival.

Exp Cell Res 2011 Apr 21;317(6):802-11. Epub 2010 Dec 21.

Institute of Medical Biology, Polish Academy of Sciences, Lodz, Poland.

Matrin 3 is an integral component of nuclear matrix architecture that has been implicated in interacting with other nuclear proteins and thus modulating the activity of proximal promoters. In this study, we evaluated the contribution of this protein to proliferation of endothelial cells. To selectively modulate matrin 3 expression, we used siRNA oligonucleotides and transfection of cells with a pEGFP-N1-Mtr3. Our data indicate that downregulation of matrin 3 is responsible for reduced proliferation and leads to necrosis of endothelial cells. This conclusion is supported by observations that reducing matrin 3 expression results in (a) producing signs of necrosis detected by PI staining, LDH release, and scatter parameters in flow cytometry, (b) affecting cell cycle progression. It does not cause (c) membrane asymmetry of cells as indicated by lack of Annexin V binding as well as (d) activation of caspase 3 and cleavage of PARP. We conclude that matrin 3 plays a significant role in controlling cell growth and proliferation, probably via formation of complexes with nuclear proteins that modulate pro- and antiapoptotic signaling pathways. Thus, degradation of matrin 3 may be a switching event that induces a shift from apoptotic to necrotic death of cells.
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http://dx.doi.org/10.1016/j.yexcr.2010.12.009DOI Listing
April 2011

Plasminogen activator inhibitor type 1 interacts with alpha3 subunit of proteasome and modulates its activity.

J Biol Chem 2011 Feb 6;286(8):6820-31. Epub 2010 Dec 6.

Institute of Medical Biology, Polish Academy of Sciences, Lodz 93-232, Poland.

Plasminogen activator inhibitor type-1 (PAI-1), a multifunctional protein, is an important physiological regulator of fibrinolysis, extracellular matrix homeostasis, and cell motility. Recent observations show that PAI-1 may also be implicated in maintaining integrity of cells, especially with respect to cellular proliferation or apoptosis. In the present study we provide evidence that PAI-1 interacts with proteasome and affects its activity. First, by using the yeast two-hybrid system, we found that the α3 subunit of proteasome directly interacts with PAI-1. Then, to ensure that the PAI-1-proteasome complex is formed in vivo, both proteins were coimmunoprecipitated from endothelial cells and identified with specific antibodies. The specificity of this interaction was evidenced after transfection of HeLa cells with pCMV-PAI-1 and coimmunoprecipitation of both proteins with anti-PAI-1 antibodies. Subsequently, cellular distribution of the PAI-1-proteasome complexes was established by immunogold staining and electron microscopy analyses. Both proteins appeared in a diffuse cytosolic pattern but also could be found in a dense perinuclear and nuclear location. Furthermore, PAI-1 induced formation of aggresomes freely located in endothelial cytoplasm. Increased PAI-1 expression abrogated degradation of degron analyzed after cotransfection of HeLa cells with pCMV-PAI-1 and pd2EGFP-N1 and prevented degradation of p53 as well as IκBα, as evidenced both by confocal microscopy and Western immunoblotting.
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http://dx.doi.org/10.1074/jbc.M110.173781DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3057828PMC
February 2011

Bomapin is a redox-sensitive nuclear serpin that affects responsiveness of myeloid progenitor cells to growth environment.

BMC Cell Biol 2010 Apr 30;11:30. Epub 2010 Apr 30.

Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.

Background: Haematopoiesis is a process of formation of mature blood cells from hematopoietic progenitors in bone marrow. Haematopoietic progenitors are stimulated by growth factors and cytokines to proliferate and differentiate, and they die via apoptosis when these factors are depleted. An aberrant response to growth environment may lead to haematological disorders. Bomapin (serpinb10) is a hematopoietic- and myeloid leukaemia-specific protease inhibitor with unknown function.

Results: We found that the majority of naturally expressed bomapin was located in the nucleus. Both the natural and recombinant bomapin had a disulfide bond which linked the only two bomapin cysteines: one located in the CD-loop and the other near the C-terminus. Computer modelling showed that the cysteines are distant in the reduced bomapin, but can easily be disulfide-linked without distortion of the overall bomapin structure. Low-level ectopic expression of bomapin in bomapin-deficient K562 cells resulted in about 90% increased cell proliferation under normal growth conditions. On the other hand, antisense-downregulation of natural bomapin in U937 cells resulted in a decreased cell proliferation. Bomapin C395S mutant, representing the reduced form of the serpin, had no effect on cell proliferation, suggesting that the disulfide bond-linked conformation of bomapin is biologically important. The bomapin-dependent effect was specific for myeloid cells, since ectopic expression of the serpin in HT1080 cells did not change cell proliferation. In contrast to the survival-promoting activity of bomapin in cells cultured under optimal growth conditions, bomapin enhanced cell apoptosis following growth factor withdrawal.

Conclusions: We propose that bomapin is a redox-sensitive nuclear serpin that augments proliferation or apoptosis of leukaemia cells, depending on growth factors availability.
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http://dx.doi.org/10.1186/1471-2121-11-30DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2874763PMC
April 2010

Autocrine effects of VEGF-D on endothelial cells after transduction with AD-VEGF-D(DeltaNDeltaC).

Exp Cell Res 2010 Apr 22;316(6):907-14. Epub 2010 Jan 22.

Department of Molecular and Medical Biophysics, Medical University of Lodz, Lodz, Poland.

Endothelial cells in tumor vessels display unusual characteristics in terms of survival and angiogenic properties which result from the increased expression of VEGF-D and its autocrine effect. To evaluate mechanisms by which VEGF-D leads to such abnormal phenotype, we searched for proteins with modified expression in HUVECs enriched in the recombinant mature VEGF-D (VEGFD(DeltaNDeltaC)) delivered by adenovirus. Expression of membrane proteins in endothelial cells was characterized by FACS using anti-human IT-Box-135 antibodies. HUVECs transduced with Ad-VEGF-D(DeltaNDeltaC) revealed markedly increased expression of proteins involved in adhesion and migration such as (a) integrins (alphaVbeta5, alpha2beta1, alpha5beta1, alphaMbeta2, alphaLbeta2), (b) matrix metalloproteinases (MMP-2, MMP-9, and MMP-14), (c) components of fibrinolytic system (PAI-1, u-PAR), and (d) CD45, CD98, CD147. Interestingly, there also were numerous proteins with significantly reduced expression, particularly among surface exposed membrane proteins. Thus, it can be concluded that to induce proangiogenic phenotype and facilitate migration of HUVECs, VEGF-D(DeltaNDeltaC) not only upregulates expression of proteins known to participate in the cell-matrix interactions but also silences some membrane proteins which could interfere with this process.
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http://dx.doi.org/10.1016/j.yexcr.2010.01.014DOI Listing
April 2010