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ABSTRACT: The majority of brain metastases originate from lung cancer, breast cancer and malignant melanoma. In order to reach the brain, parenchyma metastatic cells have to transmigrate through the endothelial cell layer of brain capillaries, which forms the morphological basis of the blood-brain barrier (BBB). The BBB has a dual role in brain metastasis formation: it forms a tight barrier protecting the central nervous system from entering cancer cells, but it is also actively involved in protecting metastatic cells during extravasation and proliferation in the brain. The mechanisms of interaction of cancer cells and cerebral endothelial cells are largely uncharacterized. Here, we provide a comprehensive review on our current knowledge about the role of junctional and adhesion molecules, soluble factors, proteolytic enzymes and signaling pathways mediating the attachment of tumor cells to brain endothelial cells and the transendothelial migration of metastatic cells. Since brain metastases represent a great therapeutic challenge, it is indispensable to understand the mechanisms of the interaction of tumor cells with the BBB in order to find targets of prevention of brain metastasis formation.
International Journal of Molecular Sciences 01/2013; 14(1):1383-411. · 2.60 Impact Factor
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István Sziráki,
Franciska Erdo,
Péter Trampus,
Mirabella Sike,
Petra Magdolna Molnár,
Zsuzsanna Rajnai,
Judit Molnár,
Imola Wilhelm, Csilla Fazakas,
Emese Kis,
István Krizbai,
Péter Krajcsi
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ABSTRACT: An integrated assay system involving dual/triple-probe microdialysis techniques in rats was developed earlier for testing interactions with P-glycoprotein (P-gp) at the blood-brain barrier using quinidine/PSC-833 as a P-gp substrate/inhibitor combination. The aim of the present study was to expand our assay system to mice using microdialysis with simultaneous sampling of blood and brain and to compare the result with a primary mouse brain endothelial cell monolayer (pMBMEC) assay. Brain penetration of quinidine was dose dependent in both anesthetized and awake mice after intraperitoneal drug administration. PSC-833 pretreatment caused a 2.5- to 3.4-fold increase in quinidine levels of brain dialysate samples in anesthetized or awake animals, after single or repeated administration of PSC-833. In pMBMEC, a 2.0- to 2.5-fold efflux ratio was observed in the transcellular transport of quinidine. The P-gp-mediated vectorial transport of quinidine was eliminated by PSC-833. These results indicate that quinidine with PSC-833 is a good probe substrate-reference inhibitor combination for testing drug-drug interactions with P-gp in the in vivo and in vitro mouse systems. With increasing number of humanized transgenic mice, a test system with mouse microdialysis experimentation becomes more important to predict drug-drug interactions in humans.
Journal of Biomolecular Screening 11/2012; · 2.05 Impact Factor
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ABSTRACT: Mechanical parameters play a crucial role in proper cellular functions. This article examines the process of the appearance and breaking of adhesion forces during contact between the confluent cerebral endothelial cell layer and a melanoma cell attached to a tipless cantilever. This adhesion is the initial phase of melanoma transmigration through the endothelial cell layer. Taking the force measurement, if the contact was prolonged for several seconds, a decrease in the load force was observed, which corresponds to stress relaxation of the cells. The dependence of adhesion force and stress relaxation on dwell time showed a saturation-like behavior. These stress relaxation curves could be fitted with the sum of two exponentials, suggesting that two independent processes take place simultaneously. The breakup of the adhesion during the retraction of the cantilever with the attached melanoma cell is not continuous but shows jumps. Between living endothelial and melanoma cells, a minimum jump size of about 20 pN could be determined. The minimum jump is independent of the dwell time and load force. It seems to be the elementary binding force between these two cell types. In case of fixed endothelial cells, the adhesion force was strongly decreased and the jumps disappeared, whereas the stress relaxation did not show considerable change upon fixation.
Biophysics of Structure and Mechanism 02/2012; 41(2):139-45. · 2.44 Impact Factor
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ABSTRACT: Because of their poor metabolic stability and limited blood-brain barrier permeability, endomorphins have a low analgesic efficacy when administered systemically. Therefore, it is of great importance to design analogues with improved peptidase resistance and better delivery to the central nervous system. Recently, novel endomorphin-2 analogues have been synthesized, which proved to bind with high affinity and selectivity to the μ-opioid receptors and showed proteolytic resistance. In this study, we have analysed the transport characteristics of endomorphin-2 and three of its analogues [Dmt-Pro-Phe-Phe-NH(2) , Tyr-(1S,2R)Acpc-Phe-Phe-NH(2) and Tyr-(1S,2R)Achc-Phe-Phe-NH(2) ] using an in vitro blood-brain barrier model. The lipophilicity of the analogues, as assessed by their octanol/water partition coefficients, was higher than that of endomorphin-2. The flux of all four peptides from the apical (blood) side to the basolateral (brain) side was not saturable in the 10nm-1mm concentration range, suggesting that a passive mechanism plays a major role in their transport. The permeability coefficient of the analogues was significantly higher than that of endomorphin-2, suggesting increased blood-brain barrier penetration properties. We conclude that because of their good peptidase resistance and improved transport through brain endothelial cells, these endomorphin-2 analogues will have better analgesic properties in vivo.
Chemical Biology & Drug Design 12/2011; 79(4):507-13. · 2.28 Impact Factor
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István Sziráki,
Franciska Erdo,
Erzsébet Beéry,
Petra Magdolna Molnár, Csilla Fazakas,
Imola Wilhelm,
Ildikó Makai,
Emese Kis,
Krisztina Herédi-Szabó,
Tibor Abonyi,
István Krizbai,
Gábor K Tóth,
Péter Krajcsi
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ABSTRACT: This study provides evidence that quinidine can be used as a probe substrate for ABCB1 in multiple experimental systems both in vitro and in vivo relevant to the blood-brain barrier (BBB). The combination of quinidine and PSC-833 (valspodar) is an effective tool to assess investigational drugs for interactions on ABCB1. Effects of quinidine and substrate-inhibitor interactions were tested in a membrane assay and in monolayer assays. The authors compared quinidine and digoxin as ABCB1 probes in the in vitro assays and found that quinidine was more potent and at least as specific as digoxin in ATPase and monolayer efflux assays employing MDCKII-MDR1 and the rat brain microcapillary endothelial cell system. Brain exposure to quinidine was tested in dual-/triple-probe microdialysis experiments in rats by assessing levels of quinidine in blood and brain. Comparing quinidine levels in dialysate samples from valspodar-treated and control animals, it is evident that systemic/local administration of the inhibitor diminishes the pumping function of ABCB1 at the BBB, resulting in an increased brain penetration of quinidine. In sum, quinidine is a good probe to study ABCB1 function at the BBB. Moreover, quinidine/PSC-833 is an ABCB1-specific substrate/inhibitor combination applicable to many assay systems both in vitro and in vivo.
Journal of Biomolecular Screening 08/2011; 16(8):886-94. · 2.05 Impact Factor
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ABSTRACT: The reliable determination of the mechanical properties of a living cell is one of the most important challenges of the atomic force microscopic measurements. In the present study the spatial and temporal dependency of the force measurements on cerebral endothelial cells was investigated. Besides imaging the cells, two different sequences of force measurements were applied: Acquisition of force curves in short time at several points across the cell surface investigating spatial dependence of the elasticity. Acquisition of force curves for long time at a previously determined place, over the cell nucleus, which provides the temporal stability/variation of the measured forces/values. Three different stages of endothelial cell cultures of the hCMEC/D3 cells were used: sub-confluent living, confluent living, and confluent fixed cells. The Young's modulus was calculated from the force curves using the Hertz model and the results were plotted against time or location correspondingly. The rational of using the three stage of culture was to clarify whether the observed effect belongs to the individual cell, to the ensemble of cells or just to some, not living cell component. In case of sub-confluent cells the results revealed a softer nuclear region compared to the periphery, while an attenuated oscillation like fluctuation in time, with a period of about 10-30 min, was observed. Confluent living cells showed similar tendencies to the sub-confluent cells, but the changes were larger and the temporal oscillations had longer period. The spatial dependency of the elasticity on confluent cells was confirmed by force-volume measurement too. In case of fixed cells neither spatial nor temporal differences were observed between the nuclear and peripheral region, however the Young's modulus and the error of the measurement was larger, compared to the sub-confluent living cells.
Journal of Molecular Recognition 04/2011; 24(3):422-8. · 3.31 Impact Factor
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ABSTRACT: The blood-brain barrier (BBB) is an active interface between the circulation and the central nervous system (CNS) with a dual function: the barrier function restricts the transport from the blood to the brain of potentially toxic or harmful substances; the carrier function is responsible for the transport of nutrients to the brain and removal of metabolites. The BBB plays a crucial role in the clinical practice as well. On the one side there is a large number of neurological disorders including cerebral ischemia, brain trauma and tumors, neurodegenerative disorders, in which the permeability of the BBB is increased. On the other hand due to the relative impermeability of the barrier many drugs are unable to reach the CNS in therapeutically relevant concentration, making the BBB one of the major impediments in the treatment of CNS disorders. The significant scientific and industrial interest in the physiology and pathology of the BBB led to the development of several in vitro models of the BBB. These models are mainly based on the culture of cerebral endothelial cells. The best in vitro models which mimic the best way the in vivo anatomical conditions are the co-culture models in which brain endothelial cells are co-cultured with astrocytes and/or pericytes. Our in vitro BBB model is characterized by high transendothelial electrical resistance (TEER regularily above 200 Ohm x cm(2)), low permeability and expression of several transporters. Our experiments have proven that the model is suitable for basic research and for testing the interaction between the BBB and potential drug candidates (toxicity, permeability, interaction with efflux transporters) as well.
Acta neurobiologiae experimentalis 01/2011; 71(1):113-28. · 2.11 Impact Factor
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Csilla Fazakas,
Imola Wilhelm,
Péter Nagyoszi,
Attila E Farkas,
János Haskó,
Judit Molnár,
Hannelore Bauer,
Hans-Christian Bauer,
Ferhan Ayaydin,
Ngo Thi Khue Dung,
László Siklós,
István A Krizbai
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ABSTRACT: Malignant melanoma represents the third common cause of brain metastasis, having the highest propensity to metastasize to the brain of all primary neoplasms in adults. Since the central nervous system lacks a lymphatic system, the only possibility for melanoma cells to reach the brain is via the blood stream and the blood-brain barrier. Despite the great clinical importance, mechanisms of transmigration of melanoma cells through the blood-brain barrier are incompletely understood. In order to investigate this question we have used an in vitro experimental setup based on the culture of cerebral endothelial cells (CECs) and the A2058 and B16/F10 melanoma cell lines, respectively. Melanoma cells were able to adhere to confluent brain endothelial cells, a process followed by elimination of protrusions and transmigration from the luminal to the basolateral side of the endothelial monolayers. The transmigration process of certain cells was accelerated when they were able to use the routes preformed by previously transmigrated melanoma cells. After migrating through the endothelial monolayer several melanoma cells continued their movement beneath the endothelial cell layer. Melanoma cells coming in contact with brain endothelial cells disrupted the tight and adherens junctions of CECs and used (at least partially) the paracellular transmigration pathway. During this process melanoma cells produced and released large amounts of proteolytic enzymes, mainly gelatinolytic serine proteases, including seprase. The serine protease inhibitor Pefabloc® was able to decrease to 44-55% the number of melanoma cells migrating through CECs. Our results suggest that release of serine proteases by melanoma cells and disintegration of the interendothelial junctional complex are main steps in the formation of brain metastases in malignant melanoma.
PLoS ONE 01/2011; 6(6):e20758. · 4.09 Impact Factor
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ABSTRACT: Cerebral endothelial cells - the principal components of the blood-brain barrier (BBB) - fulfill several important functions in the central nervous system (CNS). They form an active interface between blood and neuronal tissue and play a key role in the maintenance of the homeostasis of the CNS. Infections caused by different pathogens are often associated with systemic symptoms and may compromise the functional integrity of the BBB as well. In the mediation of the systemic effect of pathogens Toll-like receptors (TLRs) play a significant role. TLRs are a type of pattern recognition receptor and recognize molecules that are broadly shared by pathogens but distinguishable from host molecules. TLRs are broadly distributed on cells of the immune system and function as primary sensors of invading pathogens. There is also growing experimental evidence indicating that Toll-like receptors are expressed on different non-immune cell types as well, like epithelial or endothelial cells. Here we demonstrate the expression of TLR2, TLR3, TLR4 and TLR6 on rat and human cerebral endothelial cells. Oxidative stress significantly upregulated the expression of these receptors whereas TNF-alpha upregulated the expression of TLR2 and TLR3. Furthermore we have shown, that activation of TLR2/6 leads to an increased permeability which is accompanied by a downregulation of occludin and claudin-5 expression and disappearance of these tight junction proteins from the cell membrane. Changes in occludin expression and localization could be inhibited by the ERK1/2 inhibitor U0126. Our results suggest a significant role of the cerebral endothelium in mediation of the neural effects of different inflammatory processes.
Neurochemistry International 11/2010; 57(5):556-64. · 2.86 Impact Factor
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Imola Wilhelm,
Péter Nagyoszi,
Attila E Farkas,
Pierre-Olivier Couraud,
Ignacio A Romero,
Babette Weksler, Csilla Fazakas,
Ngo Thi Khue Dung,
Sándor Bottka,
Hannelore Bauer,
Hans-Christian Bauer,
István A Krizbai
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ABSTRACT: Because of the relative impermeability of the blood-brain barrier (BBB), many drugs are unable to reach the CNS in therapeutically relevant concentration. One method to deliver drugs to the CNS is the osmotic opening of the BBB using mannitol. Hyperosmotic mannitol induces a strong phosphorylation on tyrosine residues in a broad spectrum of proteins in cerebral endothelial cells, the principal components of the BBB. Previously, we have shown that among targets of tyrosine phosphorylation are beta-catenin, extracellular signal-regulated kinase 1/2 and the non-receptor tyrosine kinase Src. The aim of this study was to identify new signalling pathways activated by hypertonicity in cerebral endothelial cells. Using an antibody array and immunoprecipitation we identified the receptor tyrosine kinase Axl to become tyrosine phosphorylated in response to hyperosmotic mannitol. Besides activation, Axl was also cleaved in response to osmotic stress. Degradation of Axl proved to be metalloproteinase- and proteasome-dependent and resulted in 50-55 kDa C-terminal products which remained phosphorylated even after degradation. Specific knockdown of Axl increased the rate of apoptosis in hyperosmotic mannitol-treated cells; therefore, we assume that activation of Axl may be a protective mechanism against hypertonicity-induced apoptosis. Our results identify Axl as an important element of osmotic stress-induced signalling.
Journal of Neurochemistry 09/2008; 107(1):116-26. · 4.06 Impact Factor
