Jacek Szymański

Inducible Heat Shock Protein 70 Promotes Myelin Autoantigen Presentation by the HLA Class II

In this study, we investigated the role of the inducible form of heat shock protein 70 (hsp70) in the presentation of the major putative autoantigen in multiple sclerosis, myelin basic protein (MBP), in the context of appropriate MHC class II. By coimmunoprecipitation, we found that MBP is associated with hsp70 in APC in an ATP/ADP-dependent manner. Additionally, using confocal microscopy, hsp70 was detected in the endocytic pathway of APC, where it colocalized with MBP and HLA-DR. The immunodominant epitopes of MBP 85–99 and 80–99 were shown to bind selectively and specifically to hsp70 by surface plasmon resonance. The functional significance of MBP interaction with hsp70 was demonstrated by the detection of enhanced responses of an MBP-specific T cell hybridoma to MBP and MBP 80–99 with increasing levels of hsp70 and reduced responses when hsp70 expression was diminished within APC-expressing DRA*0101, DRB1*1501 (DR1501). However, when MBP 85–99 was used as the stimulus, T cell hybridoma responses were not enhanced by hsp70 overexpression within APC, suggesting that hsp70 contributes to Ag processing rather than Ag presentation. The importance of a direct association between MBP and hsp70 in the presentation pathways was demonstrated by enhanced efficacy of MBP presentation by APC transfected with a plasmid vector encoding a fusion hsp70-MBP protein. This is the first report on the involvement of self-inducible hsp70 in MHC class II-dependent autoantigen processing by APC. It implicates that aberrant self hsp expression may lead to the enhancement/modulation of autoimmune responses.

Interaction and functional association of protein disulfide isomerase with αVβ3 integrin on endothelial cells

Adhesive properties of endothelial cells are influenced by the thioldisulfide balance. However, the molecular mechanism of this effect is unclear, although recent observations indicate that integrin receptors may be direct targets for redox modulation. The purpose of this study was to examine whether protein disulfide isomerase (PDI) is directly involved in this process. As manganese ions are known to affect the thioldisulfide balance and activate integrins to maximal affinity, we searched for PDI interactions with integrins, particularly with αVβ3, in Mn2+‐treated endothelial cells. By employing confocal microscopy, flow cytometry and coimmunoprecipitation experiments, we showed that exposure of endothelial cells to Mn2+ resulted in: (a) the appearance of surface protein thiol groups, which can be found in PDI and αVβ3, and both proteins colocalizing on the cellular surface; and (b) the formation of the PDI–αVβ3 complex, which dissociates upon reduction. In addition, PDI in a complex with αVβ3 induces conversion of the integrin to the ligand‐competent high‐affinity state, as evidenced by increased binding of vitronectin. The membrane‐impermeable sulfhydryl blockers 3‐N‐maleimidylpropionyl biocytin 3‐N‐maleimidylpropionyl biocytin and p‐chloromercuriphenyl sulfonate, as well as the PDI inhibitors bacitracin, MA3 018, and MA3 019, abolished the binding of vitronectin and LM609 to endothelial cells that is activated by Mn2+. Consistently, LM609 almost completely blocked binding of vitronectin to such cells. The formation of the PDI–αVβ3 stoichiometric complex was further demonstrated by surface plasmon resonance analysis, which showed that the initial reversible binding of PDI becomes irreversible in the presence of Mn2+, probably mediated by disulfide bonds. Thus, we show that Mn2+ simultaneously modulates the thiol isomerase activity of PDI that is bound to αVβ3 and induces its transition to the ligand‐competent state, suggesting an alternative mechanism of integrin regulation.

Structural Rearrangements of the 10–23 DNAzyme to β3 Integrin Subunit mRNA Induced by Cations and Their Relations to the Catalytic Activity

The intracellular ability of the “10–23” DNAzyme to efficiently inhibit expression of targeted proteins has been evidenced by in vitro and in vivo studies. However, standard conditions for kinetic measurements of the DNAzyme catalytic activity in vitro include 25 mm Mg2+, a concentration that is very unlikely to be achieved intracellularly. To study this discrepancy, we analyzed the folding transitions of the 10–23 DNAzyme induced by Mg2+. For this purpose, spectroscopic analyzes such as fluorescence resonance energy transfer, fluorescence anisotropy, circular dichroism, and surface plasmon resonance measurements were performed. The global geometry of the DNAzyme in the absence of added Mg2+ seems to be essentially extended, has no catalytic activity, and shows a very low binding affinity to its RNA substrate. The folding of the DNAzyme induced by binding of Mg2+ may occur in several distinct stages. The first stage, observed at 0.5 mm Mg2+, corresponds to the formation of a compact structure with limited binding properties and without catalytic activity. Then, at 5 mm Mg2+, flanking arms are projected at right position and angles to bind RNA. In such a state, DNAzyme shows substantial binding to its substrate and significant catalytic activity. Finally, the transition occurring at 15 mm Mg2+ leads to the formation of the catalytic domain, and DNAzyme shows high binding affinity toward substrate and efficient catalytic activity. Under conditions simulating intracellular conditions, the DNAzyme was only partially folded, did not bind to its substrate, and showed only residual catalytic activity, suggesting that it may be inactive in the transfected cells and behave like antisense oligodeoxynucleotide.

Fibrinogen Contains Cryptic PAI-1 Binding Sites That Are Exposed on Binding to Solid Surfaces or Limited Proteolysis

Objective—In this work, we identified the fibrinogen sequence that on exposure serves as the primary binding site for functionally active PAI-1 and to a lesser extent for its latent form. In contrast, this site only weakly interacts with PAI-1 substrate. Methods and Results—The binding site is located in the N-terminal α (20-88) segment of fibrinogen, in the region exposed on (1) adsorption of fibrinogen to solid surfaces; (2) the release of fibrinopeptide A during thrombin conversion of fibrinogen to fibrin; and (3) plasmin degradation of fibrinogen. This region was first identified by the yeast 2-hybrid system, then its binding characteristics were evaluated using the recombinant α(16-120) fragment and its shorter version, the α(20-88) fragment, in a solid phase binding assay and plasmon surface resonance measurements. Because fibrinogen fragment E does not bind PAI-1, it suggests that sequences of Aα chain interacting with PAI-1 are located in the N-terminal part of the α(20-88) segment. Conclusions—Therefore, PAI-1 directly bound to the α(20-88) and thus concentrated in fibrinogen/fibrin, particularly at sites of injury and inflammation, may account for the recent observations that both its active and latent forms stimulate cell migration and wound healing.

Modified C-reactive protein interacts with platelet glycoprotein Ibα

Herein, we investigated the possible mechanisms by which recombinant modified CRP(m(r)CRP) modulates blood platelet function. Modified CRP could activate blood platelets and stimulate their adhesion and aggregation in the absence of any other physiological stimuli. Preincubation of isolated blood platelets with m(r)CRP at a concentration as low as 2 μg/ml resulted in significant platelet degranulation (fraction of CD62-positive platelets increased 2-fold, p < 0.0002), and at concentrations of 20 μg/ml and 100 μg/ml, increased exposure of the platelet procoagulant surface was observed (expression of annexin V-positive platelets increased to 5.7 ± 1.0% and 10.4 ± 2.2%, respectively, p < 0.03, vs. 2.9 ± 0.2% in control). Furthermore, m(r)CRP (100 μg/ml) strongly augmented spontaneous and ADP-induced fibrinogen binding to platelets (p < 0.05), platelet adhesion to fibrinogen and platelet aggregation. Using the Biacore™ surface plasmon resonance technique and glycoprotein Ibα (GPIbα) immobilized on the sensor surface, we demonstrated direct binding between platelet GPIbα and m(r)CRP. Binding of m(r)CRP to GPIbα and C1q was also observed by ELISA, irrespective of the immobilized ligand. These outcomes strongly support a role of the GPIb-IX-V complex in the interactions of m(r)CRP with blood platelets.

Anticancer Activity of New Synthetic α‐Methylene‐δ‐Lactones on Two Breast Cancer Cell Lines

Natural products are important leads in drug discovery. The search for effective plant‐derived anticancer agents or their synthetic analogues has continued to be of interest to biologists and chemists for a long time. In this report, cytotoxicity and anticancer activity of new synthetic α‐methylene‐δ‐lactones was tested against two breast cancer cell lines, invasive, hormone‐independent MDA‐MB‐231 and hormone‐dependent MCF‐7. Cytotoxicity was examined using MTT assay. The ability to induce apoptosis and changes in mitochondrial membrane potential was studied by flow cytometry. The expression levels of pro‐ and anti‐apoptotic genes were determined by quantitative real‐time PCR. Cancer cell migration and invasion were assessed by wound healing and Matrigel assays. Additionally, secretion of proteins associated with invasiveness, metalloproteinase‐9 (MMP‐9) and urokinase plasminogen activator (uPA) was investigated using commercial ELISA kits and MMP‐9 activity by gelatin zymography. A natural sesquiterpene lactone, parthenolide, was used as a positive control. Screening results showed all four analogues to be highly cytotoxic. The most potent compound of the series, 1‐isopropyl‐2‐methylene‐1,2‐dihydrobenzochromen‐3‐one, designated DL‐3, which reduced the number of viable MDA‐MB‐231 and MCF‐7 cells with the IC50 values of 5.3 μM and 3.54 μM, respectively, was selected for further research. DL‐3 activated the intrinsic pathway of apoptosis, associated with the loss of mitochondrial membrane potential and changes in Bax/Bcl‐2 ratio. DL‐3 also inhibited the movement of both types of breast cancer cells. Suppression of cell migration and invasion was the result of the decreased secretion of enzymes responsible for the degradation of the extracellular matrix, MMP‐9 and uPA. These findings show that the synthetic α‐methylene‐δ‐lactone, DL‐3, displays potential to be further explored in the development of new anticancer agents.

New cyclopentaquinoline derivatives with fluorobenzoic acid induce G1 arrest and apoptosis in human lung adenocarcinoma cells.

Non-small cell lung cancer accounts for 80-85% of all lung cancer cases and is the leading cause of cancer death indicating inefficient current treatment. Acridine derivatives interact with DNA and inhibit topoisomerase leading to cell growth arrest or cell death. The aim of this study was to evaluate the effects of new synthesized sixteen 2,3-dihydro-1H-cyclopenta[b]quinoline derivatives (cyclopentaquinoline), a member of acridine-based compounds, on the survival and growth of human lung adenocarcinoma, A549 cells. Anticancer activity of eight new cyclopentaquinoline derivatives with hydrazinonicotinic acid (compounds 1-8) and eight with fluorobenzoic acid (compounds 9-16) were screened using WST-1 assay. Interestingly, cyclopentaquinoline derivatives with fluorobenzoic moiety were found to have a higher anticancer activity than derivatives with hydrazinonicotinic acid. Four out of eight tested compounds with fluorobenzoic acid inhibited 50% cancer cell growth at concentration below 20μM. Moreover, the efficacy of cyclopentaquinoline derivatives containing fluorobenzoic acid correlated with increasing number of carbon atoms in the aliphatic chain. The most effective compounds (6, 15, 16) were selected to determine molecular mechanisms of their anticancer action. The results indicated that inhibition of A549 cell growth by compounds 15 and 16 was associated with a cell cycle arrest at G0/1 phase and with induction of caspase 3-dependent apoptosis. Compound 6 also caused A549 cells death due to apoptosis, however, it had no significant effect on a cell cycle progression. These findings suggest that cyclopentaquinoline derivatives containing fluorobenzoic acid with 8 and 9 carbon atoms in aliphatic chain may be promising candidate for treatment of lung cancer.

Novel tetrahydroacridine derivatives inhibit human lung adenocarcinoma cell growth by inducing G1 phase cell cycle arrest and apoptosis

Lung cancer is not only the most commonly diagnosed cancers worldwide but it is still the leading cause of cancer-related death. Acridine derivatives are a class of anticancer agents with the ability to intercalate DNA and inhibit topoisomerases. The aim of this study was to evaluate the effect of sixteen new tetrahydroacridine derivatives on the viability and growth of human lung adenocarcinoma cells. We compared anticancer activity of a series of eight compounds with 4-fluorobenzoic acid and eight compounds with 6-hydrazinonicotnic acid differed from each other in length of the aliphatic chain containing from 2 to 9 carbon atoms. Interestingly, tetrahydroacridine with 4-fluorobenzoic acid (compounds 9-16) showed higher anticancer activity than derivatives with 6-hydrazinonicotnic acid (compounds 1-8) and their efficacy was correlated with increasing number of carbon atoms in the aliphatic chain. The results showed that inhibition of cancer cell growth by the most effective compounds 15 and 16 was associated with induction of G1 phase cell cycle arrest followed by caspase-3 dependent apoptosis. Our findings suggest that tetrahydroacridine with 4-fluorobenzoic acid containing 8 and 9 carbon atoms may be potential candidate for treatment of lung cancer.

Flavanols from Japanese quince (Chaenomeles japonica) fruit suppress expression of cyclooxygenase-2, metalloproteinase-9, and nuclear factor-kappaB in human colon cancer cells

Natural polyphenols and polyphenol-rich extracts have been found to possess preventive and therapeutic potential against several types of cancers, including colorectal cancer (CRC), which is an example of an inflammation-associated cancer. This study examines the chemopreventive effect of a Japanese quince (Chaenomeles japonica) fruit flavanol preparation (JQFFP) on colon cancer SW-480 cells. JQFFP, rich in procyanidin monomers and oligomers, was found to inhibit the SW-480 cell viability by 40% at 150 µM catechin equivalents (CE) after 72 h incubation when compared to control, but it was non-toxic to normal colon fibroblast CCD-18Co cells. Furthermore, 100 µM CE JQFFP suppressed COX-2 mRNA expression to 36.7% of control values and protein expression to 77%. In addition, JQFFP reduced the MMP-9 protein expression (to 24% vs. control at 100 µM CE) and caused inhibition of its enzymatic activity (to 35% vs. control at 100 µM CE). Not only did JQFFP inhibit the COX-2 and MMP-9 levels, but it also reduced the NF-κB protein expression (to 65% of control) and phosphorylation of its p65 subunit (to 51%) at 100 µM CE. These results provide the first evidence that JQFFP inhibits COX-2, MMP-9, and NF-κB expression, suggesting that it has cytotoxic, anti-inflammatory, and anti-metastatic activities towards the colon cancer SW-480 cells.

The Behavior of Embryonic Neural Cells within the 3D Micro-structured Collagen-Based Scaffolds

Synthesis of scaffolds providing mechanical support for the growing cells is important in reconstruction of the tissue. Addition of chondroitin sulfate (CS) to collagen scaffolds was proved to regulate the neural cells growth and differentiation. The aim of the study is to test whether collagen-CS cross-linked scaffolds could be used for embryonic neuronal cell culture. Embryonic neuronal cells were isolated from rat foetus brains and their differentiation into neurons or astrocytes was characterized by flow cytometry. Then, the entrapment and distribution of cells within collagen alone or composite collagen with CS scaffolds was studied. The cells were applied to the scaffold and stained with bisbenzimide and then counted. Finally the MTT test was performed. The results suggest that embryonic neuronal cells were differentiated into neurons (MAP2 positive cells) or astrocytes (GFAP positive cells). The cells entered into the two tested 3D scaffolds; however their distribution in the tested scaffolds was not homogenous. The MTT test showed more intensive metabolism of the cells in collagen with CS scaffolds comparing with controls (cells seeded on laminin) or cells growing in scaffolds composed only from cross-linked collagen. Conclusion: the both collagen and collagen with CS scaffolds are the good carriers for the embryonic neuronal cells. However, the sample composed of collagen with CS constitutes better conditions for embryonic neuronal cells culture.