Publications by authors named "Joanna Rossowska"

47 Publications

Monosubstituted Acetophenone Thiosemicarbazones as Potent Inhibitors of Tyrosinase: Synthesis, Inhibitory Studies, and Molecular Docking.

Pharmaceuticals (Basel) 2021 Jan 18;14(1). Epub 2021 Jan 18.

Department of Bioorganic Chemistry, Wrocław University of Science and Technology, 50-370 Wrocław, Poland.

A set of 12 monosubstituted acetophenone thiosemicarbazone derivatives (TSCs) were synthesized and their inhibitory properties toward tyrosinase activity were tested. Moreover, their ability to inhibit melanogenesis in the B16F10 murine melanoma cell line was studied. In order to investigate the nature of interactions between the enzyme and the inhibitors, molecular docking to the active site was performed. TSCs 5, 6, 8, and 9 revealed a half maximal inhibitory concentration (IC) below 1 µM. Compound 6 turned out to be the most potent tyrosinase inhibitor. All investigated compounds showed reversible inhibition of competitive or mixed type. The -substituted TSCs had higher affinity for the enzyme as compared to their - and -analogues. All investigated compounds inhibited melanin production in B16F10 cells at the micromolar level. Molecular docking showed that the sulfur atom of the thiourea moiety penetrates the active site and interacts with copper ions. The above outcomes might be helpful in the design of new tyrosinase inhibitors in the food and cosmetic industries.
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http://dx.doi.org/10.3390/ph14010074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7831505PMC
January 2021

Investigating the photodynamic efficacy of chlorin e6 by millisecond pulses in metastatic melanoma cells.

Bioelectrochemistry 2021 Apr 29;138:107728. Epub 2020 Dec 29.

Department of Physical and Quantum Chemistry, Faculty of Chemistry, Wroclaw University of Science and Technology, Poland.

Melanoma is considered the most aggressive type of skin cancer, still without effective treatment. Thus, alternative therapeutic methods are still in demand, and electroporation-supported photodynamic therapy (EP-PDT) of cancer cells seems a promising approach. New developments in EP-PDT aim at enhanced tumor selectivity and biocompatibility by applying a second-generation photosensitizer, i.e., Chlorin e6 (Ce6). We have verified the improved photodynamic effect of Ce6 on skin cancer melanoma (Me45) cells and control (CHO-K1) cells. In this study, we applied 1 or 5 pulses of 10 ms duration and assessed the EP-PDT effect with a variety of tests, such as singlet oxygen scavenger (ABMDMA) photooxidation, oxidoreductive potential measurements, kinetic measurements with fluorescent microscopy, photosensitizer uptake studies, lipid peroxidation level, and actin fibers organization. The optimization of photosensitizer uptake as a function of temperature was also performed. Our results indicated efficient Ce6 delivery into Me45 cells and good photodynamic efficiency enhanced by the electroporation of cancer cells.
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http://dx.doi.org/10.1016/j.bioelechem.2020.107728DOI Listing
April 2021

Immunomodulatory potential of anticancer therapy composed of methotrexate nanoconjugate and dendritic cell‑based vaccines in murine colon carcinoma.

Oncol Rep 2021 Mar 11;45(3):945-962. Epub 2021 Jan 11.

Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 53‑114 Wroclaw, Poland.

Chemotherapy with low‑molecular weight compounds, despite elimination of cancer cells, entails adverse effects. To overcome this disadvantage, innovative drug delivery systems are being developed, including conjugation of macromolecular carriers with therapeutics, e.g. a nanoconjugate of hydroxyethyl starch and methotrexate (HES‑MTX). The purpose of the present study was to determine whether HES‑MTX, applied as a chemotherapeutic, is able to modulate the immune response and support the antitumor response generated by dendritic cells (DCs) used subsequently as immunotherapeutic vaccines. Therefore, MTX or HES‑MTX was administered, as sole treatment or combined with DC‑based vaccines, to MC38 colon carcinoma tumor‑bearing mice. Alterations in antitumor immune response were evaluated by multiparameter flow cytometry analyses and functional assays. The results demonstrated that the nanoconjugate possesses greater immunomodulatory potential than MTX as reflected by changes in the landscape of immune cells infiltrating the tumor and increased cytotoxicity of splenic lymphocytes. In contrast to MTX, therapy with HES‑MTX as sole treatment or combined with DC‑based vaccines, contributed to significant tumor growth inhibition. However, only treatment with HES‑MTX and DC‑based vaccines activated the systemic specific antitumor response. In conclusion, due to its immunomodulatory properties, the HES‑MTX nanoconjugate could become a potent anticancer agent used in both chemo‑ and chemoimmunotherapeutic treatment schemes.
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http://dx.doi.org/10.3892/or.2021.7930DOI Listing
March 2021

Cepharanthine induces ROS stress in glioma and neuronal cells via modulation of VDAC permeability.

Saudi Pharm J 2020 Nov 4;28(11):1364-1373. Epub 2020 Sep 4.

Department of Molecular and Cellular Biology, Faculty of Pharmacy, Wroclaw Medical University, Wroclaw, Poland.

Cepharanthine (CEP) is a bisbenzylisoquinoline alkaloid. Molecular dynamics studies show that CEP interacts with Voltage-dependent anion channel (VDAC), inducing the voltage-independent channel narrowing. In the new conformation, transport between mitochondria and cytoplasm is altered, which leads to the dose-dependent cytotoxicity. The biological effects of the interaction were investigated on glioblastoma multiforme (SNB-19) and neuronal (PC-12 + NGF) cell lines. The cytotoxic potential of cepharanthine was determined by MTT assay and flow cytometry apoptosis/necrosis studies. T-type calcium channel and VDAC were labelled by the immunocytochemical method. Additionally, fluorescent labelling of reactive oxygen species and mitochondria was performed. Changes in the pore size of VDAC were calculated as well. Molecular dynamics simulations were carried out to examine the interactions of cepharanthine with VDAC. The obtained results prove that cepharanthine enhances the apoptosis in glioma and neuronal cells by the release of reactive oxygen species. Cepharanthine alters the mitochondria-to-cytoplasm transport and thus induces the cytotoxicity with no selectivity.
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http://dx.doi.org/10.1016/j.jsps.2020.08.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7679435PMC
November 2020

Protein Disulphide Isomerase A1 Is Involved in the Regulation of Breast Cancer Cell Adhesion and Transmigration via Lung Microvascular Endothelial Cells.

Cancers (Basel) 2020 Oct 2;12(10). Epub 2020 Oct 2.

Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, 30-348 Krakow, Poland.

Cancer cell cross-talk with the host endothelium plays a crucial role in metastasis, but the underlying mechanisms are still not fully understood. We studied the involvement of protein disulphide isomerase A1 (PDIA1) in human breast cancer cell (MCF-7 and MDA-MB-231) adhesion and transendothelial migration. For comparison, the role of PDIA1 in proliferation, migration, cell cycle and apoptosis was also assessed. Pharmacological inhibitor, bepristat 2a and PDIA1 silencing were used to inhibit PDIA1. Inhibition of PDIA1 by bepristat 2a markedly decreased the adhesion of breast cancer cells to collagen type I, fibronectin and human lung microvascular endothelial cells. Transendothelial migration of breast cancer cells across the endothelial monolayer was also inhibited by bepristat 2a, an effect not associated with changes in ICAM-1 expression or changes in cellular bioenergetics. The silencing of PDIA1 produced less pronounced anti-adhesive effects. However, inhibiting extracellular free thiols by non-penetrating blocker p-chloromercuribenzene sulphonate substantially inhibited adhesion. Using a proteomic approach, we identified that β1 and α2 integrins were the most abundant among all integrins in breast cancer cells as well as in lung microvascular endothelial cells, suggesting that integrins could represent a target for PDIA1. In conclusion, extracellular PDIA1 plays a major role in regulating the adhesion of cancer cells and their transendothelial migration, in addition to regulating cell cycle and caspase 3/7 activation by intracellular PDIA1. PDIA1-dependent regulation of cancer-endothelial cell interactions involves disulphide exchange and most likely integrin activation but is not mediated by the regulation of ICAM-1 expression or changes in cellular bioenergetics in breast cancer or endothelial cells.
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http://dx.doi.org/10.3390/cancers12102850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7601413PMC
October 2020

3PO as a Selective Inhibitor of 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 3 in A375 Human Melanoma Cells.

Anticancer Res 2020 May;40(5):2613-2625

Department of Molecular and Cellular Biology, Wroclaw Medical University, Wroclaw, Poland

Background/aim: The occurrence of BRAF mutation causes an up-regulation of the B-raf kinase activity leading to the stabilization of hypoxia-inducible factor 1-alpha (HIF-1α) - the promoter of the 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) enzyme. The aim of the study was to examine the effect of the (2E)-3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), as an inhibitor of PFKFB3, on human melanoma cells (A375) with endogenous BRAF mutation.

Materials And Methods: A375 cells were exposed to different concentrations of 3PO and the following tests were performed: docking, cytotoxicity assay, immunocytochemistry staining glucose uptake, clonogenic assay, holotomography imaging, and flow cytometry.

Results: Our studies revealed that 3PO presents a dose-dependent and time-independent cytotoxic effect and promotes apoptosis of A375 cells. Furthermore, the obtained data indicate that 3PO induces cell cycle arrest in G1/0 and glucose uptake reduction.

Conclusion: Taking all together, our research demonstrated a here should be proapoptotic and antiproliferative effect of 3PO on A375 human melanoma cells.
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http://dx.doi.org/10.21873/anticanres.14232DOI Listing
May 2020

Halogenated aromatic thiosemicarbazones as potent inhibitors of tyrosinase and melanogenesis.

Bioorg Chem 2020 01 9;94:103419. Epub 2019 Nov 9.

Department of Bioorganic Chemistry, Wrocław University of Science and Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland. Electronic address:

A set of 21 halogenated thiosemicarbazones (TSCs) have been synthesized and its inhibitory properties toward activity diphenolase of mushroom tyrosinase and their ability to inhibition of melanogenesis in B16F10 murine, melanoma cell line have been investigated. The molecular docking to the active site of the enzyme has been also performed to investigate the nature of enzyme-inhibitor interactions. The obtained outcomes allowed us to perform SAR analysis. TSC 6, 12 and 21 exhibited the most potent inhibitory properties showing IC of 0.5, 0.9 and 0.8 µM, respectively. They revealed reversible and competitive manner of tyrosinase inhibition. According to SAR analysis, para-substituted acetophenone derivatives of thiosemicarbazones have the highest affinity to the enzyme among the investigated compounds. Melanin production in B16F10 cells was inhibited by all investigated compounds at the micromolar level. Suggested inhibition mechanism is based on the interaction between a sulfur atom of thiourea moiety of the thiosemicarbazones, and copper ions in the active site of the enzyme. These results might be useful in searching novel inhibitors of melanogenesis which could be used in the cosmetic and food industry.
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http://dx.doi.org/10.1016/j.bioorg.2019.103419DOI Listing
January 2020

De‑O‑acylated lipooligosaccharide of E. coli B reduces the number of metastatic foci via downregulation of myeloid cell activity.

Oncol Rep 2020 Jan 8;43(1):270-281. Epub 2019 Nov 8.

Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 53‑114 Wroclaw, Poland.

Lipopolysaccharides are the main surface antigens and virulence factors of gram‑negative bacteria. Removal of four ester‑bound fatty acid residues from hexaacyl lipid A of Escherichia coli lipooligosaccharide (LOS) resulted in the de‑O‑acylated derivative E. coli LOS‑OH (LOS‑OH). This procedure caused a significant reduction in the toxicity of this compound compared to the native molecule. We investigated the effect of such a structural LOS modification on its biological activity using in vitro assays with monocytic cells of the RAW264.7 line, dendritic cells of the JAWS II line, bone marrow‑derived dendritic cells (BM‑DCs), and spleen cells. Furthermore, in in vivo experiments with a melanoma B16 metastasis model, the anti‑metastatic activity of the compounds and spleen cell reactivity mediated by them representing a systemic response were analyzed. The results revealed that LOS‑OH demonstrated weaker ability than LOS to stimulate and polarize an immune response both in vitro and in vivo. It induced lower cytokine production by cells of myeloid lines. Multiple applications of LOS‑OH into mice injected intravenously with B16 cells significantly (P<0.05; P<0.01) reduced the number of metastatic foci in the lungs, presumably via silencing of myeloid cell reactivity as well as the inability to stimulate lymphoid cells both directly and indirectly. These findings suggest that LOS‑OH maintained in the body of metastasis‑bearing mice appears to modulate or downregulate the innate response, leading to the inability of blood myeloid cells to support the migration of melanoma cells to lung tissue.
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http://dx.doi.org/10.3892/or.2019.7403DOI Listing
January 2020

Effects of Photosensitization of Curcumin in Human Glioblastoma Multiforme Cells.

In Vivo 2019 Nov-Dec;33(6):1857-1864

Department of Molecular and Cellular Biology, Wroclaw Medical University, Wroclaw, Poland

Background/aim: There is no satisfactory treatment of glioblastoma multiforme, a highly invasive brain tumor. The aim of this study was to analyze the cytotoxic effects of curcumin (CUR) alone and as a photosensitizer on glioblastoma cells.

Materials And Methods: The SNB-19 cells where incubated for 2 and 24 h with 5-200 mM of CUR. The cells were radiated with blue light (6 J/cm) and compared to non-irradiated ones. The effects of treatment were assessed by measuring mitochondrial activity with the MTT method and apoptosis progression by flow cytometry. To investigate CUR uptake, fluorescence imaging of cells was performed.

Results: Photosensitization of CUR decreased the EC 6.3 times when the incubation time was 2 h and over 90% of cells underwent apoptosis. The study of the uptake of CUR showed that during the 2 h, CUR was placed in the entire cytoplasm, and over time, its amount decreased and localized in the subcellular compartments.

Conclusion: CUR is a promising medicament that can be used as a photosensitizer in photodynamic therapy for glioma treatment.
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http://dx.doi.org/10.21873/invivo.11679DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6899084PMC
March 2020

The Cytoprotective Role of Antioxidants in Mammalian Cells Under Rapidly Varying UV Conditions During Stratospheric Balloon Campaign.

Front Pharmacol 2019 2;10:851. Epub 2019 Aug 2.

Department of Molecular and Cellular Biology, Wroclaw Medical University, Wroclaw, Poland.

The current age of dynamic development of the space industry brings the mankind closer to routine manned space flights and space tourism. This progress leads to a demand for intensive astrobiological research aimed at improving strategies of the pharmacological protection of the human cells against extreme conditions. Although routine research in space remains out of our reach, it is worth noticing that the unique severe environment of the Earth's stratosphere has been found to mimic subcosmic conditions, giving rise to the opportunity to use the stratospheric surface as a research model for the astrobiological studies. Our study included launching into the stratosphere a balloon containing mammalian normal and cancer cells treated with various compounds to examine whether these substances are capable of protecting the cells against stress caused by rapidly varying temperature, pressure, and radiation, especially UV. Owing to oxidative stress caused by irradiation and temperature shock, we used natural compounds which display antioxidant properties, namely, catechin isolated from green tea, honokiol derived from magnolia, curcumin from turmeric, and cinnamon extract. "After-flight" laboratory tests have shown the most active antioxidants as potential agents which can minimize harmful impact of extreme conditions on human cells.
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http://dx.doi.org/10.3389/fphar.2019.00851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6687761PMC
August 2019

Tacalcitol increases the sensitivity of colorectal cancer cells to 5-fluorouracil by downregulating the thymidylate synthase.

J Steroid Biochem Mol Biol 2019 06 25;190:139-151. Epub 2019 Mar 25.

Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 12 Rudolfa Weigla, 53-114, Wroclaw, Poland.

5-Fluorouracil (5-FU) is an anticancer drug that is most frequently used to treat colorectal cancer (CRC) patients, but unfortunately it shows limited efficacy. We recently demonstrated that vitamin D analogs (VDAs), particularly tacalcitol (coded as PRI-2191), potentiate its anticancer activity in an in vivo mouse and human CRC model. The purpose of this study was to explain the mechanism underlying the enhancement of 5-FU efficacy by PRI-2191 towards human HT-29 CRC cells. We showed that PRI-2191 induces the CDKN1A (gene encoding p21) expression directly through vitamin D receptor (VDR) in a p53-independent manner and thus decreases the thymidylate synthase expression both at the mRNA and protein level. It is the main mechanism by which PRI-2191 improves the anticancer efficacy of 5-FU towards HT-29 cells. Additionally, we indicated that the VDR also participates in 5-FU mechanism of action. 5-FU significantly increased TYMS (gene encoding thymidylate synthase (TS)) and BIRC5 (gene encoding survivin) level in HT-29 cells with silenced VDR. Furthermore, PRI-2191 induced E-cadherin and ZO-1 expression and thus reduced the level of BIRC5 in HT-29 cells. The induction of E-cadherin expression may also contribute to the reduction of c-Myc level and consequently the downregulation of TS. Our results also indicate that calcium-sensing receptor (CaSR) plays a role in the activity of PRI-2191 but has no influence on the 5-FU mechanism of action. In conclusion, we suggest that both VDR and CaSR might be useful as molecular markers for predicting treatment outcomes and identifying the CRC patient subgroups who might benefit from 5-FU-based chemotherapy combined with vitamin D analog.
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http://dx.doi.org/10.1016/j.jsbmb.2019.03.017DOI Listing
June 2019

Antitumor Potential of Extracellular Vesicles Released by Genetically Modified Murine Colon Carcinoma Cells With Overexpression of Interleukin-12 and shRNA for TGF-β1.

Front Immunol 2019 13;10:211. Epub 2019 Feb 13.

Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland.

Recent developments demonstrate that tumor-derived extracellular vesicles (EVs) could become a highly effective tool for delivery of antitumor factors. The main objective of the study was to determine whether EVs secreted by MC38 colon carcinoma cells genetically engineered for overproduction of interleukin (IL-)12 and/or shRNA targeting TGF-β1 are effectively loaded with these molecules and whether the obtained EVs could be an efficient tool for antitumor therapy. Fractions of EVs released by genetically modified MC38 cells [both modified tumor-derived exosomes (mTEx) and modified microvesicles (mTMv)] and those released by unmodified, wild-type MC38 cells were characterized in terms of loading efficacy, using real-time PCR and ELISA, as well as their antitumor potential. In order to examine the therapeutic potential of mTEx, they were applied in the form of sole treatment as well as in combination with dendritic cell (DC)-based vaccines stimulated with mTMv in the therapy of mice with subcutaneously growing MC38 tumors. The results demonstrated that genetic modification of wild-type MC38 tumor cells is an effective method of loading the molecules of interest into extracellular vesicles secreted by the cells (both TEx and TMv). The results also showed that mTEx secreted by cells engineered for overproduction of IL-12 and/or shRNA for TGF-β1 are able to induce tumor growth inhibition as opposed to TEx from unmodified MC38 cells. Additionally, antitumor therapy composed of mTEx (especially those deprived of TGF-β1) and DC-based vaccines allowed for regeneration of antitumor immunity and induction of the systemic Th1 response responsible for the sustained effect of the therapy. In conclusion, tumor-derived exosomes loaded with IL-12 and/or deprived of TGF-β1 could become an efficient adjuvant supporting induction of a specific antitumor response in both immuno- and chemotherapeutic schemes of treatment.
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http://dx.doi.org/10.3389/fimmu.2019.00211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6381037PMC
January 2020

Reprogramming the murine colon cancer microenvironment using lentivectors encoding shRNA against IL-10 as a component of a potent DC-based chemoimmunotherapy.

J Exp Clin Cancer Res 2018 Jun 28;37(1):126. Epub 2018 Jun 28.

Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. R. Weigla 12, 53-114, Wroclaw, Poland.

Background: The excessive amounts of immunosuppressive factors present in a tumor microenvironment (TME) reduce the effectiveness of cancer vaccines. The main objective of our research was to improve the effectiveness of dendritic cell (DC)-based immunotherapy or chemoimmunotherapy composed of cyclophosphamide (CY) and DCs by application of lentivectors encoding shRNA specific to IL-10 (shIL10 LVs) in murine colon carcinoma MC38 model.

Methods: The efficacy of shIL10 LVs in silencing of IL-10 expression was measured both in vitro and in vivo using Real-Time PCR and ELISA assays. In addition, the influence of intratumorally inoculated lentivectors on MC38 tumor microenvironment was examined using flow cytometry method. The effect of applied therapeutic schemes was determined by measurement of tumor growth inhibition and activation state of local and systemic immune response.

Results: We observed that intratumorally inoculated shIL10 LVs transduced tumor and TME-infiltrating cells and reduced the secretion of IL-10. Application of shIL10 LVs for three consecutive weeks initiated tumor growth inhibition, whereas treatment with shIL10 LVs and BMDC/TAg did not enhance the antitumor effect. However, when pretreatment with CY was introduced to the proposed scheme, we noticed high MC38 tumor growth inhibition accompanied by reduction of MDSCs and Tregs in TME, as well as activation of potent local and systemic Th1-type antitumor response.

Conclusions: The obtained data shows that remodeling of TME by shIL10 LVs and CY enhances DC activity and supports them during regeneration and actuation of a potent antitumor response. Therefore, therapeutic strategies aimed at local IL-10 elimination using lentiviral vectors should be further investigated in context of combined chemoimmunotherapies.
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http://dx.doi.org/10.1186/s13046-018-0799-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6025815PMC
June 2018

The favourable effect of catechin in electrochemotherapy in human pancreatic cancer cells.

Acta Biochim Pol 2018 22;65(2):173-184. Epub 2018 May 22.

Department of Medical Biochemistry, Wroclaw Medical University, Wrocław, Poland.

Until recently, green tea polyphenols were considered strong antioxidants. However, the latest reports have revealed that bioflavonoids can play a multiple role in anticancer therapy, including the inhibition of cell proliferation and generation of the oxidative stress in a dose-dependent manner. The presented research was designed to examine the potential of the green tea (±)-catechin as a reinforcement of the electrochemotherapy (ECT) with cisplatin in pancreatic cancer in vitro. The study was performed on two cell lines of the pancreatic ductal adenocarcinoma (PDA) - parental EPP85-181P and multidrug-resistant EPP85-181RNOV. Prior to the ECT protocol the cells were preincubated with high or low concentration of catechin for 2 or 24 hours, respectively. We assessed the influence of preincubation on the cisplatin toxicity with and without electroporation (EP), the electrosensitivity of PDA cell lines and the uptake of the daunorubicin and propidium iodide. Additionally, we evaluated the antioxidative properties of catechin by the measurement of the ROS-related fluorescence and the immunoreactivity of the oxidative stress-related enzymes superoxide dismutase (SOD2) and glutathione S-transferase (GST). We found that co-treatment with catechin can firmly enhance the efficacy of electroporation with cisplatin in vitro. More favorable effect was obtained for 2-hour incubation, which indicates the involvement of the transcriptional-independent mechanisms of catechin action. The effect may be partially explained by the increased oxidative stress level, which was higher in multidrug-resistant cells. However, further studies on cisplatin-catechin interplay and the thorough examination of the catechin-cell membrane interaction need to be performed.
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http://dx.doi.org/10.18388/abp.2018_2602DOI Listing
October 2018

Electroporation with Cisplatin against Metastatic Pancreatic Cancer: Study on Human Primary Cell Culture.

Biomed Res Int 2018 19;2018:7364539. Epub 2018 Mar 19.

Department of Thoracic Surgery, Wroclaw Medical University, Wroclaw, Poland.

Despite the rapid progression of cancer pharmacotherapy, the high drug resistance of pancreatic ductal adenocarcinoma (PDA) makes it one of the most lethal malignancies. Therefore, there are high expectations associated with experimental therapies, such as electrochemotherapy (ECT). This technique involves the application of short electric pulses to induce transitional permeabilization of the cellular membrane, thus enhancing drug molecules influx. The aim of the study was to investigate the influence of electroporation with cisplatin (CisEP) on the primary culture of human PDA cells from lung metastases-their survival and stress response. Considering the growing importance of various research models, two established human PDA cell lines, EPP85-181P (sensitive to daunorubicin) and EPP85-181RDB (resistant to daunorubicin), were utilized as a reference control. Cisplatin revealed higher cytotoxicity towards established cell lines. Following CisEP application, we observed a significant decrease of cells viability in the primary culture model. After CisEP therapy, an increased immunoreactivity with SOD-2 and Casp-3 antibodies was noticed. In conclusion, we discovered that electroporation can enhance the cytotoxic effect of cisplatin in pancreatic cancer cells . This effect was evident for cells from the primary culture. The obtained results confirm the importance of primary cells models in studies on the efficacy of experimental cancer therapies.
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http://dx.doi.org/10.1155/2018/7364539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5884438PMC
September 2018

Biological effects in photodynamic treatment combined with electropermeabilization in wild and drug resistant breast cancer cells.

Bioelectrochemistry 2018 Oct 19;123:9-18. Epub 2018 Apr 19.

Faculty of Fundamental Problems of Technology, Wrocław University of Science and Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland. Electronic address:

Electrochemotherapy became one of the therapeutic protocols successfully used in oncology. However, biological effects occurring in cells, especially those which are drug resistant, have not been studied thoroughly. This study presents response of wild and drug resistant breast cancer cells to classical photodynamic therapy with Photofrin or experimental photodynamic therapy with cyanine IR-775, combined with electroporation. Photodynamic reaction or electroporation alone had no cytotoxic effect, but their combination significantly disturbed cellular functions. Applying electroporation allowed the drugs to increase its accumulation, especially for a poorly permeant cyanine in drug resistant cells. FACS analysis showed that even at relatively mild electric field, ca. 90% of cells were permeabilized. High intracellular concentration of drugs triggered the cellular defense system through increased expression of glutathione S-transferase and multidrug resistance proteins (MDR1 and MRP7), particularly in drug resistant cells. Finally, expressively decreased cell metabolism and proliferation, as well as formation of apoptotic bodies and fragmentation of cells were observed after the combined treatment. The results show that electroporation can be used for effective delivery of photosensitizers, even to drug resistant breast cancer cells, which was not tested before. This shows that electro-photodynamic treatment could be a promising approach to overcome a problem of drug resistance in cancer cells.
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http://dx.doi.org/10.1016/j.bioelechem.2018.04.008DOI Listing
October 2018

The role of immune cell subpopulations in the growth and rejection of TC-1/A9 tumors in novel mouse strains differing in the H2-D haplotype and NKC domain.

Oncol Lett 2018 Mar 10;15(3):3594-3601. Epub 2018 Jan 10.

Department of Health Care Disciplines and Population Protection, Czech Technical University in Prague, Faculty of Biomedical Engineering, 27201 Kladno, Czech Republic.

The present study aimed to elucidate the role of cluster of differentiation (CD)8+, CD4+, natural killer (NK), and myeloid (CD11b+) cells in the course of the growth and rejection of experimental major histocompatibility complex (MHC) class I-deficient, HPV16 E6/E7-associated TC-1/A9 tumors in mice. Stable mouse lines (F) generated by inbreeding of Balb/c and C57BL/6 strains, which were characterized by H-2Db+d-NK1.1neg (B6-neg) and H-2Db-d+NK1.1high (Balb-high) phenotypes, were used for the present study. The novel strains spontaneously regressed tumors in 70-90% of cases. histological analysis of the tumor microenvironment in cryosections showed an indirect correlation between the growth of the transplanted tumor (progressor vs. regressor mice) and the proportion of immunocompetent cell infiltration in the tumors. The regressor mice exhibited a higher infiltration of tumors with CD4+ and CD8+ cells, and in Balb-high with NK cells as well, compared with the progressors. All tumor transplants also indicated a huge infiltration of CD11b+ cells, but this infiltration was not dependent on the stage of the TC-1/A9 tumor development. Depletion of individual cell subpopulations exhibited different effects on the tumor development in the two strains. Elimination of CD8-positive cells enhanced growth of TC-1/A9 tumor transplants in both hybrid stains, whereas CD4+ cell depletion affected rejection of TC-1/A9 tumors in the B6-neg mice only. Depletion of NK cells with anti-asialo GM1 antibody in the Balb-high strain led to enhancement of tumor growth, which was more pronounced after depletion of the NK1.1+ subpopulation. On the other hand, depletion of NK cells with anti-asialo GM1 in B6-neg mice did not affect the regression of TC-1/A9 tumor transplants, but increased the CD11b+ cell infiltration. In summary, these results indicate that co-operation of particular subsets of immunocompetent cells is essential for the rejection of TC-1/A9 tumor transplants. In B6-neg mice, the co-operative action of CD8+ and CD4+ cells is required, whereas in Balb-high mice, the synergy of CD8+ and NK1.1+ cells is of major importance.
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http://dx.doi.org/10.3892/ol.2018.7763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5795941PMC
March 2018

Immunological landscape of consensus clusters in colorectal cancer.

Oncotarget 2017 Dec 27;8(62):105299-105311. Epub 2017 Oct 27.

Department of Genetics, Wroclaw Medical University, Wroclaw, Poland.

Recent, large-scale expression-based subtyping has advanced our understanding of the genomic landscape of colorectal cancer (CRC) and resulted in a consensus molecular classification that enables the categorization of most CRC tumors into one of four consensus molecular subtypes (CMS). Currently, major progress in characterization of immune landscape of tumor-associated microenvironment has been made especially with respect to microsatellite status of CRCs. While these studies profoundly improved the understanding of molecular and immunological profile of CRCs heterogeneity less is known about repertoire of the tumor infiltrating immune cells of each CMS. In order to comprehensively characterize the immune landscape of CRC we re-analyzed a total of 15 CRC genome-wide expression data sets encompassing 1597 tumors and 125 normal adjacent colon tissues. After quality filtering, CRC clusters were discovered using a combination of multiple clustering algorithms and multiple validity metrics. CIBERSORT algorithm was used to compute relative proportions of 22 human leukocyte subpopulations across CRC clusters and normal colon tissue. Subsequently, differential expression specific to tumor epithelial cells was calculated to characterize mechanisms of tumor escape from immune surveillance occurring in particular CRC clusters. Our results not only characterize the common and cluster-specific influx of immune cells into CRCs but also identify several deregulated gene targets that may contribute to improvement of immunotherapeutic strategies in CRC.
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http://dx.doi.org/10.18632/oncotarget.22169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5739639PMC
December 2017

Cell Membrane Transport Mechanisms: Ion Channels and Electrical Properties of Cell Membranes.

Adv Anat Embryol Cell Biol 2017 ;227:39-58

Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS, 205 Route de Narbonne, BP 64182, F-31077, Toulouse, France.

Cellular life strongly depends on the membrane ability to precisely control exchange of solutes between the internal and external (environmental) compartments. This barrier regulates which types of solutes can enter and leave the cell. Transmembrane transport involves complex mechanisms responsible for passive and active carriage of ions and small- and medium-size molecules. Transport mechanisms existing in the biological membranes highly determine proper cellular functions and contribute to drug transport. The present chapter deals with features and electrical properties of the cell membrane and addresses the questions how the cell membrane accomplishes transport functions and how transmembrane transport can be affected. Since dysfunctions of plasma membrane transporters very often are the cause of human diseases, we also report how specific transport mechanisms can be modulated or inhibited in order to enhance the therapeutic effect.
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http://dx.doi.org/10.1007/978-3-319-56895-9_3DOI Listing
May 2018

MRP1 protein expression in leukemic stem cells as a negative prognostic marker in acute myeloid leukemia patients.

Eur J Haematol 2017 Nov 29;99(5):415-422. Epub 2017 Sep 29.

Department and Clinic of Hematology, Blood Neoplasms and Bone Marrow Transplantation, Wroclaw Medical University, Wroclaw, Poland.

Background: It is well established that expression of multi-drug resistance (MDR) proteins (MDR1, BCRP, MDR3, MRP1, and LRP) in leukemic blasts correlates with acute myeloid leukemia (AML) patients' clinical response. Assuming that leukemic stem cells (LSC) are resistant to chemotherapy and responsible for relapse, it might be clinically relevant to evaluate the expression level of MDR proteins in LSC and relate it to the clinical outcome.

Methods: Bone marrow samples from 26 patients with de novo AML were labeled with antibodies to distinguish CD34+CD38-CD123+ LSC population and with antibodies against MDR1, BCRP, MDR3, MRP1, or LRP proteins. Multicolor flow cytometry was applied to evaluate the expression of MDR proteins in blasts and LSC.

Results: Nine of 26 patients with AML attained CR (30%). High negative correlation was found between MDR1 and LRP expression in blasts and the patient's remission. MDR proteins were expressed more frequently in LSC than in leukemic blasts. High negative correlation was also observed between remission achievement and MRP1 expression in LSC.

Conclusions: Our data present for the very first time the high negative correlation between MRP1 protein expression in LSC and AML patients' remission. It does strongly suggest that MRP1 expression in LSC is an adverse prognostic marker in patients with de novo AML.
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http://dx.doi.org/10.1111/ejh.12938DOI Listing
November 2017

Intratumoral Lentivector-Mediated TGF-β1 Gene Downregulation As a Potent Strategy for Enhancing the Antitumor Effect of Therapy Composed of Cyclophosphamide and Dendritic Cells.

Front Immunol 2017 30;8:713. Epub 2017 Jun 30.

Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland.

Vaccination with dendritic cells (DCs) stimulated with tumor antigens can induce specific cellular immune response that recognizes a high spectrum of tumor antigens. However, the ability of cancer cells to produce immunosuppressive factors drastically decreases the antitumor activity of DCs. The main purpose of the study was to improve the effectiveness of DC-based immunotherapy or chemoimmunotherapy composed of cyclophosphamide (CY) and DCs by application of lentivectors (LVs)-encoding short hairpin RNA specific for TGF-β1 (shTGFβ1 LVs). We observed that s.c. inoculation of both MC38 cells with silenced expression of TGF-β1 (MC38/shTGF-β1) and direct intratumoral application of shTGFβ1 LVs contributed to reduction of suppressor activity of myeloid cells and Tregs in tumor. Contrary to expectations, in mice bearing wild tumor, the application of shTGFβ1 LVs prior to vaccination with bone marrow-derived DC stimulated with tumor antigens (BMDC/TAg) did not influence myeloid-derived suppressor cell (MDSC) infiltration into tumor. As a result, we observed only minor MC38 tumor growth inhibition (TGI) accompanied by systemic antitumor response activation comparable to that obtained for negative control (shN). However, when the proposed scheme was complemented by pretreatment with a low dose of CY, we noticed high MC38 TGI together with decreased number of MDSCs in tumor and induction of Th1-type response. Moreover, in both schemes of treatment, LVs (shTGFβ1 as well as shN) induced high influx of CTLs into tumor associated probably with the viral antigen introduction into tumor microenvironment. Concluding, the application of shTGFβ1 LVs alone or in combination with DC-based vaccines is not sufficient for long-lasting elimination of suppression in tumor. However, simultaneous reduction of TGF-β1 in tumor microenvironment and its remodeling by pretreatment with a low dose of CY facilitates the settlement of peritumorally inoculated DCs and supports them in restoration and activation of a potent antitumor response.
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http://dx.doi.org/10.3389/fimmu.2017.00713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5492852PMC
June 2017

Differentiated all-trans retinoic acid response of naive CD4+CD25- cells isolated from rats with collagen-induced arthritis and healthy ones under conditions.

Cent Eur J Immunol 2017 8;42(1):39-53. Epub 2017 May 8.

Department of Immunology, Pathophysiology, and Prevention, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Science, Wroclaw, Poland.

Aim O The Study: To compare the potential of CD4+CD25- cells, isolated from both healthy rats and rats with CIA (Collagen-Induced Arthritis), for differentiation into regulatory T cells in the presence of all-trans retinoic acid in order to learn more about the activation mechanisms and therapeutic potential of regulatory T cells.

Material And Methods: Sorted CD4+CD25- cells were cultured in vitro with/without ATRA, and then the frequency of regulatory T cells and their ability to secrete IL-10 by CD4+ FOXP3+ cells was examined. Gene expression of the foxp3, rarα, rarβ, rxrβ, and ppar β/δ and protein expression of the Rarα, Rarβ, and Rxrβ in cells after stimulation with ATRA were also investigated.

Results: CD4+CD25- cells isolated from healthy animals or from animals with CIA are characterised by different potential of the differentiation into CD4+CD25+ FOXP3+ cells. Retinoic acid receptor Rxrβ is present in the CD4+CD25- cells isolated from rats with CIA.

Conclusions: We showed that although ATRA did not increase the frequency of Treg in culture, it significantly increased expression of rarβ and rxrβ only in lymphocytes taken from diseased animals and foxp3 expression only in healthy animals. Moreover, after ATRA stimulation, the frequency of Treg-produced IL-10 tended to be lower in diseased animals than in the healthy group. The results imply that the potential of naïve cell CD4 lymphocytes to differentiate into Tregs and their putative suppressive function is dependent on the donor's health status.
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http://dx.doi.org/10.5114/ceji.2017.67317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5470613PMC
May 2017

Polyelectrolyte Oil-Core Nanocarriers for Localized and Sustained Delivery of Daunorubicin to Colon Carcinoma MC38 Cells: The Case of Polysaccharide Multilayer Film in Relation to PEG-ylated Shell.

Macromol Biosci 2017 05 17;17(5). Epub 2017 Jan 17.

Department of Organic and Pharmaceutical Technology, Faculty of Chemistry, Wroclaw University of Science and Technology, Wybrzeze Wyspianskiego 27, 50-370, Wroclaw, Poland.

The authors examine properties of daunorubicin (DNR)-loaded oil-core multilayer nanocapsules prepared via layer-by-layer approach with different polyelectrolyte (PE) coatings such as a standard one (containing polysodium 4-styrenesulphonate/poly(diallyldimethyl-ammonium) chloride) and a polysaccharide-based shell (dextran/chitosan), in regard to the outer layer of poly-l-glutamic acid (PGA) grafted with polyethylene glycol (PGA-g-PEG). The nanocarriers are obtained on a cationic nanoemulsion template (stabilized by dicephalic-type surfactant, N,N-bis[3,30-(trimethylammonio)propyl]-dodecanamide dimethylsulfate) and layered with the PE shell of different thicknesses resulting in average size of 150 nm in diameter (as shown by dynamic light scattering, scanning electron microscopy and cryogenic-transmission electron microscopy, and atomic force microscopy). The nanocapsules demonstrate efficient DNR encapsulation and its sustained release under physiological conditions or in the attendance of human serum albumin. The biocompatibility studies using colon carcinoma MC38 and macrophage P388D1 cell lines as well as human erythrocytes reveal that surface charge and outer PE layer type determine nanocarrier features that control their biological activity: protein adsorption, cellular internalization and localization, induction of apoptosis, and hemolytic activity. The investigations indicate that polysaccharide-coated nanocapsules present a considerable potential for application as efficient DNR delivery systems in chemotherapy of colon cancer as an alternative to nanocarriers with PEG-ylated shell.
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http://dx.doi.org/10.1002/mabi.201600356DOI Listing
May 2017

Structural and immunomodulatory differences among lactobacilli exopolysaccharides isolated from intestines of mice with experimentally induced inflammatory bowel disease.

Sci Rep 2016 11 21;6:37613. Epub 2016 Nov 21.

L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wrocław, Poland.

Characteristic changes in the microbiota biostructure and a decreased tolerance to intestinal bacteria have been associated with inflammatory bowel disease (IBD). However, few studies have examined the constituents of the intestinal microbiota, including the surface molecules of the bacteria, in healthy and IBD subsets. Here, we compare the chemical structures and immunomodulatory properties of the exopolysaccharides (EPS) of lactobacilli isolated from mice with induced IBD (IBD "+") versus those of healthy mice (IBD "-"). Classical structural analyses were performed using nuclear magnetic resonance spectroscopy and mass spectrometry. Immunomodulatory properties were assessed by stimulation of dendritic cells derived from mouse bone marrow or human peripheral mononuclear blood cells. Our results revealed that EPS produced by IBD "+" species are structurally different from those isolated from IBD "-". Moreover, the structurally different EPS generate different immune responses by dendritic cells. We speculate that resident strains could, upon gut inflammation, switch to producing EPS with specific motifs that are absent from lactobacilli IBD "-", and/or that bacteria with a particular EPS structure might inhabit the inflamed intestinal mucosa. This study may shed light on the role of EPS in IBD and help the development of a specific probiotic therapy for this disease.
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http://dx.doi.org/10.1038/srep37613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5116648PMC
November 2016

The Effect of Millisecond Pulsed Electric Fields (msPEF) on Intracellular Drug Transport with Negatively Charged Large Nanocarriers Made of Solid Lipid Nanoparticles (SLN): In Vitro Study.

J Membr Biol 2016 10 12;249(5):645-661. Epub 2016 May 12.

Department of Biomedical Engineering, Faculty of Fundamental Problems of Technology, Wroclaw University of Science and Technology, Wybrzeże Wyspiańskiego 27, 50-370, Wroclaw, Poland.

Drug delivery technology is still a dynamically developing field of medicine. The main direction in nanotechnology research (nanocarriers, nanovehicles, etc.) is efficient drug delivery to target cells with simultaneous drug reduction concentration. However, nanotechnology trends in reducing the carrier sizes to several nanometers limit the volume of the loaded substance and may pose a danger of uncontrolled access into the cells. On the other hand, nanoparticles larger than 200 nm in diameter have difficulties to undergo rapid diffusional transport through cell membranes. The main advantage of large nanoparticles is higher drug encapsulation efficiency and the ability to deliver a wider array of drugs. Our present study contributes a new approach with large Tween 80 solid lipid nanoparticles SLN (i.e., hydrodynamic GM-SLN-glycerol monostearate, GM, as the lipid and ATO5-SLNs-glyceryl palmitostearate, ATO5, as the lipid) with diameters DH of 379.4 nm and 547 nm, respectively. They are used as drug carriers alone and in combination with electroporation (EP) induced by millisecond pulsed electric fields. We evaluate if EP can support the transport of large nanocarriers into cells. The study was performed with two cell lines: human colon adenocarcinoma LoVo and hamster ovarian fibroblastoid CHO-K1 with coumarin 6 (C6) as a fluorescent marker for encapsulation. The biological safety of the potential treatment procedure was evaluated with cell viability after their exposure to nanoparticles and EP. The EP efficacy was evaluated by FACS method. The impact on intracellular structure organization of cytoskeleton was visualized by CLSM method with alpha-actin and beta-tubulin. The obtained results indicate low cytotoxicity of both carrier types, free and loaded with C6. The evaluation of cytoskeleton proteins indicated no intracellular structure damage. The intracellular uptake and accumulation show that SLNs do not support transport of C6 coumarin. Only application of electroporation improved the transport of encapsulated and free C6 into both treated cell lines.
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http://dx.doi.org/10.1007/s00232-016-9906-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5045845PMC
October 2016

Preparation and characterization of new zinc(II) phthalocyanine - Containing poly(l-lactide)-b-poly(ethylene glycol) copolymer micelles for photodynamic therapy.

J Photochem Photobiol B 2016 Jul 16;160:185-97. Epub 2016 Apr 16.

Department of Organic and Pharmaceutical Technology, Faculty of Chemistry, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland. Electronic address:

Poly(l-lactide)-b-poly(ethylene oxide) block copolymer (mPEG-b-PLLA) micelles were fabricated and applied as a new biodegradable and biocompatible nanocarrier for solubilization of hydrophobic zinc (II) phthalocyanine (ZnPc). The nanocarrier demonstrated a good colloidal stability and its in vitro sustained cargo release profile was assessed. Photobleaching of ZnPc, both in its native form and encapsulated in the obtained polymeric micelles, was studied by means of spectroscopic measurements. The photodynamic reaction (PDR) protocol for cyto- and photocytotoxicity was performed on metastatic melanoma cells (Me45), normal human keratinocytes (HaCaT) being used for comparison. The intracellular accumulation of free and encapsulated ZnPc was visualized at various time periods (1, 3 and 24h). The proapoptotic potential of the encapsulated phthalocyanine was evaluated by monitoring DNA double strand break damage fragmentation (TUNEL assay) and caspase 3/7 activity. In addition, in vitro biocompatibility studies were conducted by determining hemolytic activity of Zn-Pc-loaded mPEG-b-PLLA micelles and their lack of cytotoxicity against macrophages (P388/D1) and endothelial cells (HUV-EC-C). Our results suggest that the PDR using Zn-Pc-loaded mPEG-b-PLLA micelles can be effective in inhibiting tumor cell growth and apoptosis induction with higher responses, observed for Me45 cells. Additionally, the ZnPc-loaded micelles appear to be hemato-biocompatible and safe for normal keratinocytes, macrophages and endothelial cells.
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http://dx.doi.org/10.1016/j.jphotobiol.2016.04.018DOI Listing
July 2016

Electroporation and lipid nanoparticles with cyanine IR-780 and flavonoids as efficient vectors to enhanced drug delivery in colon cancer.

Bioelectrochemistry 2016 Aug 27;110:19-31. Epub 2016 Feb 27.

Department of Organic and Pharmaceutical Technology, Faculty of Chemistry, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland.

Nanocarriers and electroporation (also named electropermeabilization) are convenient methods to increase drug transport. In the current study, we present an effective support of drug delivery into cancer cells, utilizing these methods. We compare the efficiency of each of them and their combination. Multifunctional solid lipid nanoparticles (SLNs) loaded with a cyanine-type IR-780 - acting as a diagnostic agent and a photosensitizer, and a flavonoid derivative - baicalein (BAI) or fisetin (FIS) as a therapeutic cargo - were fabricated via solvent-diffusion method. A therapy supplemented with flavonoids may provide a more precise method to apply desirable lower drug doses and is more likely to result in lower toxicity and a decrease in tumor growth. The SLNs were stabilized with Phospholipon 90G at various concentrations; cetyl palmitate (CP) was applied as a solid matrix. The obtained nanosystems were characterized by dynamic light scattering (size along with size distribution), UV-vis (cargos encapsulation efficiency) and atomic force microscopy (morphology and shape). The obtained SLNs were used as drug carriers alone and in combination with electropermeabilization induced by millisecond pulsed electric fields of high intensity. Two cell lines were selected for the study: LoVo and CHO-K1. The viability was assessed after electroporation alone, the use of electroporation and nanoparticles, and nanoparticles or drugs alone. The intracellular accumulation of cyanine IR-780 and the impact on intracellular structure organization of cytoskeleton was visualized with confocal microscopy method with alpha-actin and beta-tubulin. In this study, the efficacy of nanoparticles with mixed cargo, additionally enhanced by electroporation, is demonstrated to act as an anticancer modality to eliminate cancer cells.
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http://dx.doi.org/10.1016/j.bioelechem.2016.02.013DOI Listing
August 2016

Oral Administration of Polymyxin B Modulates the Activity of Lipooligosaccharide E. coli B against Lung Metastases in Murine Tumor Models.

PLoS One 2016 1;11(2):e0148156. Epub 2016 Feb 1.

Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland.

Introduction: Polymyxin B (PmB) belongs to the group of cyclic peptide antibiotics, which neutralize the activity of LPS by binding to lipid A. The aim of this study was to analyze the effect of PmB on the biological activity of lipooligosaccharide (LOS E. coli B,rough form of LPS) in vitro and in experimental metastasis models.

Results: Cultures of murine macrophage J774A.1 cells and murine bone marrow-derived dendritic cells (BM-DC) stimulated in vitro with LOS and supplemented with PmB demonstrated a decrease in inflammatory cytokine production (IL-6, IL-10, TNF-α) and down-regulation of CD40, CD80, CD86 and MHC class II molecule expression. Additionally, PmB suspended in drinking water was given to the C57BL/6 mice seven or five days prior to the intravenous injection of B16 or LLC cells and intraperitoneal application of LOS. This strategy of PmB administration was continued throughout the duration of the experiments (29 or 21 days). In B16 model, statistically significant decrease in the number of metastases in mice treated with PmB and LOS (p<0.01) was found on the 14th day of the experiments, whereas the most intensive changes in surface-antigen expression and ex vivo production of IL-6, IL-1β and TNF-α by peritoneal cells were observed 7 days earlier. By contrast, antigen expression and ex vivo production of IL-6, IL-10, IFN-γ by splenocytes remained relatively high and stable. Statistically significant decrease in LLC metastases number was observed after the application of LOS (p<0.01) and in the group of mice preconditioned by PmB and subsequently treated with LOS (LOS + PmB, p<0.01).

Conclusions: In conclusion, prolonged in vivo application of PmB was not able to neutralize the LOS-induced immune cell activity but its presence in the organism of treated mice was important in modulation of the LOS-mediated response against the development of metastases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0148156PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4735115PMC
July 2016

Treatment with cyclophosphamide supported by various dendritic cell-based vaccines induces diversification in CD4⁺ T cell response against MC38 colon carcinoma.

Int J Oncol 2016 Feb 7;48(2):493-505. Epub 2015 Dec 7.

Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 53-114 Wroclaw, Poland.

The present study shows that an application of cyclophosphamide (CY) supported by dendritic cell (DC)-based vaccines affected differentiation of the activity of CD4+ T cell subpopulations accompanied by an alteration in CD8+ cell number. Vaccines were composed of bone marrow-derived DCs activated with tumor cell lysate (BM-DC/TAgTNF-α) and/or genetically modified DCs of JAWS II line (JAWS II/Neo or JAWS II/IL-2 cells). Compared to untreated or CY-treated mice, the combined treatment of MC38 colon carcinoma-bearing mice resulted in significant tumor growth inhibition associated with an increase in influx of CD4+ and CD8+ T cells into tumor tissue. Whereas, the division of these cell population in spleen was not observed. Depending on the nature of DC-based vaccines and number of their applications, both tumor infiltrating cells and spleen cells were able to produce various amount of IFN-γ, IL-4 and IL-10 after mitogenic ex vivo stimulation. The administration of CY followed by BM-DC/TAgTNF-α and genetically modified JAWS II cells, increased the percentage of CD4+T-bet+ and CD4+GATA3+ cells and decreased the percentage of CD4+RORγt+ and CD4+FoxP3+ lymphocytes. However, the most intensive response against tumor was noted after the ternary treatment with CY + BM-DC/TAgTNF-α + JAWS II/IL-2 cells. Thus, the administration of various DC-based vaccines was responsible for generation of the diversified antitumor response. These findings demonstrate that the determination of the size of particular CD4+ T cell subpopulations may become a prognostic factor and be the basis for future development of anticancer therapy.
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http://dx.doi.org/10.3892/ijo.2015.3278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4725454PMC
February 2016

Adoptively transferred Tregs accumulate in a site-specific manner and ameliorate signs of less advanced collagen-induced arthritis progress in rats.

Immunotherapy 2015 ;7(3):215-28

Wroclaw University of Environmental & Life Science, Faculty of Veterinary Medicine, Department of Immunology, Pathophysiology & Prevention Veterinary, Norwida 31, 50-375 Wroclaw, Poland.

Aim: The aim of the study was to assess the therapeutic effect and migration of adoptively transferred Tregs in the course of collagen-induced arthritis (CIA) in rats.

Methods: Sorted CD4(+)CD25(+) cells were cultured in the presence of 17-β-estradiol, stained with CellTracker and then administered into the articular capsule of ankle joint of animals in different stages of CIA progression.

Results: Tregs diminished CIA signs only in animals with less advanced disease progress. Moreover, migration of transferred cells into the LN in the near proximity of the injection site and with distal location was almost completely stopped in animals with fully developed CIA.

Conclusion: Disease progression-related differences in migratory potential of in vitro induced Tregs may be responsible for the failure of cellular therapy during the advanced stages of CIA.
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http://dx.doi.org/10.2217/imt.14.121DOI Listing
January 2016