Agytumorok kezelése hipoxiás sejtekre célzottan ható génterápiás módszerekkel egér tumor modellen = Treatment of brain tumors by selective targeting of hypoxic, radioresistant tumor cells in a mouse tumor model

A projekt célja a sugárrezisztens és különösen a hipoxiás daganatsejtek sugárterápiával szembeni érzékenységének fokozása volt génterápiás módszerekkel. Két sugárérzékenyítő citosztatikum hatását kívántuk génterápiás módszerrel fokozni. Először a gemcitabin toxikus és sugárérzékenyítő hatását fokoztuk úgy, hogy a daganatsejtekben túltermeltettük a gemcitabin aktivációjáért felelős deoxicitidin kináz (dCK) enzimet egy dCK-t kódoló adenovírus vektornak a daganatsejtekbe való juttatásával. Kimutattuk, hogy a különböző glioma sejtvonalak alap dCK expressziója, illetve gemcitabin érzékenysége igen eltérő és a kettő között nem találtunk összefüggést. A fokozott dCK expresszió jelentős mértékben javította mind a gemcitabin toxikus, mind annak sugárérzékenyítő hatását in vitro és in vivo, de a hatás mértéke sejt-specifikus volt. A második rendszerben a tirapazamin (TPZ) hipoxiás sugárérzékenyítő szer hatását kívántuk fokozni úgy, hogy a TPZ aktivációjáért felelős NADPH-citokróm P450 reduktáz (N-CPR) enzim génjét termeltettük túl a daganatsejtekkel. A TPZ-nek csak mérsékelt citotoxikus és sugárérzékenyítő hatása volt normoxiás körülmények között, ami hipoxiában szignifikánsan megemelkedett. Az N-CPR fokozott expressziója tovább javította a TPZ-nek a hipoxia-specifikus toxicitását és sugárérzékenyítését, mind in vitro, mind in vivo. Eredményeinkkel igazoltuk, hogy a génterápiás módszerek alkalmazásával a kombinált daganatterápia célzottabbá és hatékonyabbá tehető. | The aim of the project was to increase the radisensitivity of radioresistant and hipoxic tumor cells by the means of gene therapy. We proposed to increase the effect of two radiosensitizing chemotherapeutic agents. First, we increased the toxic and radiosensitizing effect of gemcitabine by overexpressing the deoxicitidine kinase (dCK) enzyme, responsible for the intracellular activation of the drug. For this, a dCK-encoding adenoviral vector was constructed. There was a large variation both in the basal dCK level and gemcitabine sensitivity of various glioma cell lines, and no correlation could be shown between the two of them. However, dCK overexpression could improve both the toxic and radiosensitizing effect of gemcitabine in vitro and in vivo, but the effect was cell-type specific. Next, we constructed an adenoviral vector encoding for the gene of NADPH-cytochrome-P450 reductase (N-CPR), the enzyme responsible for the intracellular activation of the hypoxic cytotoxin, tirapazamine (TPZ). TPZ had only a mild toxic and radiosensitizing effect in normoxic conditions, which was significantly increased under hypoxic conditions. N-CPR overexpression further improved the hypoxia-specific toxicity and radiosensitization of TPZ both in vitro and in vivo. Thus, our work shows that the selectivity and efficiency of the combined antitumor therapy can be improved by the means of gene therapy

A kis dózisú ionizáló sugárzás biológiai hatásai, az egyéni érzékenység szerepe a sugárhatásban. = Biological effects of low dose ionizing radiation, the effect of individual radiation susceptibility

Jelenleg közel 100, különböző sugárérzékenységű, daganatos betegek bőrbiopsziás mintáiból kialakított primer fibroblaszt kultúrával rendelkezünk. Több primer sejtkultúrát sikerült immortalizálnunk az emberi telomeráz gén retrovírus vektor segítségével történő bevitelével. Kiválasztott fibroblaszt kultúrákban a teljes emberi genomot lefedő mikroarrayvel tanulmányoztuk a 2 Gy gamma sugárzás hatására kialakuló gén-expressziós változásokat. Megállapítottuk, hogy mindössze 30 olyan gén található, amely minden fibroblaszt sejtben válaszol sugárhatásra. Az ionizáló sugárzás kis dózisok hatására kialakuló genom instabilitást kiváltó és bystander hatását tanulmányozva megállapítottuk, hogy az nincs összefüggésben a sejtek sugárérzékenységével. Bystander sejtek gén-expressziós mintázatát tanulmányozva megállapítottuk, hogy 655, 406, 152 és 619 gén működése változott meg 500, 100, 40 és 10 mGy sugár dózis hatására. 15 olyan gént találtunk, amely minden dózis hatására megváltoztatta működését bystander sejtekben. Kimutattuk, hogy a TP53INP1 gén mind közvetlen sugártalálatot szenvedett, mind pedig bystander sejtekben aktiválódik. A kis sugárdózisok jelentős mértékben befolyásolhatják az immunrendszer működését. Megállapítottuk, hogy az immunrendszer egyes sejtjei relatíve sugárérzékenyek (CD8+), míg mások sugárrezisztensek (Treg sejtek). Már 100 mGy alatti sugárdózisok is befolyásolhatják az antigén prezentáló dendritikus sejtek működését. | We have established nearly 100 primary human fibroblast cell cultures with different radiation sensitivity from skin biopsies taken from cancer patients. Several primary cultures were immortalized by the introduction of the human telomerase gene into the cells by retroviral vectors. In selected fibroblast cultures we have investigated the radiation induced transcriptional responses after 2 Gy gamma radiations by whole genome microarrys. We have detected only 30 genes which responded to radiation in all investigated cell lines. By investigating low dose radiation-induced genome instability and bystander effect in primary fibroblasts, we have found no correlation with the radiation sensitivity of the cells. Transcriptional responses were investigated in bystander cells after irradiation with 500, 100, 40 and 10 mGy. Two hours after irradiation 655, 406, 152 and 619 genes responded to these doses, respectively. Fifteen genes responded to radiation at all applied doses. Interestingly, TP53INP1 was activated both in directly irradiated and bystander cells. Low dose irradiation can seriously affect the immune system. We have found that certain lymphocyte subpopulations are relative radiation sensitive (CD8+ cells), while others are relative radiation resistant (Treg cells). The antigen presenting capacity of dendritic cells can be influenced by doses lower than 100 mGy

The synthesis of pharmacologically important oxindoles via the asymmetric aldol reaction of isatin and the investigation of the organocatalytic activity of new alicyclic β-amino acid derivatives

This work involves the synthesis and subsequent development of a number of novel organocatalysts generated from β-amino acids bearing diendo and diexo norbornene skeletons to improve their catalytic characteristics

Specific Syndecan-1 Domains Regulate Mesenchymal Tumor Cell Adhesion, Motility and Migration

Malignant mesothelioma is an asbestos induced cancer that is difficult to diagnose. Several studies have combined biomarkers to improve mesothelioma diagnosis, but with moderate success, and there is a need for new mesothelioma biomarkers. The tumour is often resistant to treatment and most patients will survive less than a year. An indicator of patient survival is the tumours growth pattern, which in turn is influenced by expressed proteoglycans. In this thesis work, we aim to improve the possibilities to diagnose malignant mesothelioma by combining biomarkers and by identifying new ones. We also investigate tumour driving mechanisms with focus on one of these suggested biomarkers, the cell-bound proteoglycan syndecan-1. We were able to construct a diagnostic two-step model based on biomarkers in patient material. By implementing a cut-off level and thereafter focusing on unresolved patients we combined hyaluronan and N-ERC/mesothelin (paper I), which significantly increased the diagnostic accuracy for malignant mesothelioma. To further improve diagnosis, we used mass spectrometry to find new biomarkers. We identified and validated galectin-1, which was excellent in discriminating mesotheliomas from adenocarcinomas (paper II). In the same study, we were also the first to describe aldo-keto reductase 1B10 as a novel prognostic mesothelioma biomarker. Syndecan-1 has been indicated as a marker for carcinomas. In paper I we describe how higher levels of syndecan-1 indicate the presence of a carcinoma over a mesothelioma. This was verified in paper II when syndecan-1 was identified as downregulated in fluids from mesothelioma patients compared to lung cancer patients. Paper III and paper IV focus on this proteoglycan. Malignant cell lines transfected with syndecan-1 and various truncated forms of syndecan-1 affected adhesion and migration, which are key features of cancer invasion (paper III). The results showed a domain- and cell type specific effect on the cells’ motility. Regulating syndecan-1 levels and analysing the global gene expression of mesothelioma cells made it evident that this proteoglycan has a strong influence on transforming growth factor β signalling and several growth factor pathways (paper IV). Links to cell migration and proliferation were furthermore identified, along with glycosaminoglycan modifying enzymes. These results can shed light on the complex role of syndecan-1 in invasion and growth of malignant mesenchymal cells. Taken together, this thesis work describes a complement to conventional mesothelioma diagnosis and identifies novel biomarkers. Furthermore, the potential biomarker syndecan-1 was shown to have an effect on cell motility and proliferation. These results increase our understanding of this aggressive malignancy

New approach methodologies to enhance human health risk assessment of immunotoxic properties of chemicals: a PARC (Partnership for the Assessment of Risk from Chemicals) project

As a complex system governing and interconnecting numerous functions within the human body, the immune system is unsurprisingly susceptible to the impact of toxic chemicals. Toxicants can influence the immune system through a multitude of mechanisms, resulting in immunosuppression, hypersensitivity, increased risk of autoimmune diseases and cancer development. At present, the regulatory assessment of the immunotoxicity of chemicals relies heavily on rodent models and a limited number of Organisation for Economic Co-operation and Development (OECD) test guidelines, which only capture a fraction of potential toxic properties. Due to this limitation, various authorities, including the World Health Organization and the European Food Safety Authority have highlighted the need for the development of novel approaches without the use of animals for immunotoxicity testing of chemicals. In this paper, we present a concise overview of ongoing efforts dedicated to developing and standardizing methodologies for a comprehensive characterization of the immunotoxic effects of chemicals, which are performed under the EU-funded Partnership for the Assessment of Risk from Chemicals (PARC)

New approach methodologies to enhance human health risk assessment of immunotoxic properties of chemicals — a PARC (Partnership for the Assessment of Risk from Chemicals) project

As a complex system governing and interconnecting numerous functions within the human body, the immune system is unsurprisingly susceptible to the impact of toxic chemicals. Toxicants can influence the immune system through a multitude of mechanisms, resulting in immunosuppression, hypersensitivity, increased risk of autoimmune diseases and cancer development. At present, the regulatory assessment of the immunotoxicity of chemicals relies heavily on rodent models and a limited number of Organisation for Economic Co-operation and Development (OECD) test guidelines, which only capture a fraction of potential toxic properties. Due to this limitation, various authorities, including the World Health Organization and the European Food Safety Authority have highlighted the need for the development of novel approaches without the use of animals for immunotoxicity testing of chemicals. In this paper, we present a concise overview of ongoing efforts dedicated to developing and standardizing methodologies for a comprehensive characterization of the immunotoxic effects of chemicals, which are performed under the EU-funded Partnership for the Assessment of Risk from Chemicals (PARC)

Ionizing Radiation-Induced Immune and Inflammatory Reactions in the Brain

Radiation-induced late brain injury consisting of vascular abnormalities, demyelination, white matter necrosis, and cognitive impairment has been described in patients subjected to cranial radiotherapy for brain tumors. Accumulating evidence suggests that various degrees of cognitive deficit can develop after much lower doses of ionizing radiation, as well. The pathophysiological mechanisms underlying these alterations are not elucidated so far. A permanent deficit in neurogenesis, chronic microvascular alterations, and blood–brain barrier dysfunctionality are considered among the main causative factors. Chronic neuroinflammation and altered immune reactions in the brain, which are inherent complications of brain irradiation, have also been directly implicated in the development of cognitive decline after radiation. This review aims to give a comprehensive overview on radiation-induced immune alterations and inflammatory reactions in the brain and summarizes how these processes can influence cognitive performance. The available data on the risk of low-dose radiation exposure in the development of cognitive impairment and the underlying mechanisms are also discussed

Extracellular Vesicles in Modifying the Effects of Ionizing Radiation

Extracellular vesicles (EVs) are membrane-coated nanovesicles actively secreted by almost all cell types. EVs can travel long distances within the body, being finally taken up by the target cells, transferring information from one cell to another, thus influencing their behavior. The cargo of EVs comprises of nucleic acids, lipids, and proteins derived from the cell of origin, thereby it is cell-type specific; moreover, it differs between diseased and normal cells. Several studies have shown that EVs have a role in tumor formation and prognosis. It was also demonstrated that ionizing radiation can alter the cargo of EVs. EVs, in turn can modulate radiation responses and they play a role in radiation-induced bystander effects. Due to their biocompatibility and selective targeting, EVs are suitable nanocarrier candidates of drugs in various diseases, including cancer. Furthermore, the cargo of EVs can be engineered, and in this way they can be designed to carry certain genes or even drugs, similar to synthetic nanoparticles. In this review, we describe the biological characteristics of EVs, focusing on the recent efforts to use EVs as nanocarriers in oncology, the effects of EVs in radiation therapy, highlighting the possibilities to use EVs as nanocarriers to modulate radiation effects in clinical applications

Syndecan-1 in Cancer: Implications for Cell Signaling, Differentiation, and Prognostication

Syndecan-1, a cell surface heparan sulfate proteoglycan, is critically involved in the differentiation and prognosis of various tumors. In this review, we highlight the synthesis, cellular interactions, and the signalling pathways regulated by syndecan-1. The basal syndecan-1 level is also crucial for understanding the sequential changes involving malignant transformation, tumor progression, and advanced or disseminated cancer stages. Moreover, we focus on the cellular localization of this proteoglycan as cell membrane anchored and/or shed, soluble syndecan-1 with stromal or nuclear accumulation and how this may carry different, highly tissue specific prognostic information for individual tumor types