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Intercellular interactions in skin tumors.
Kučera, Jan ; Smetana, Karel (advisor) ; Masařík, Michal (referee) ; Kovář, Marek (referee)
The dissertation is focused on the study of intercellular interactions in skin tumors. It is based on 5 original publications that cover several topics. We studied the origin of tumor-associated fibroblasts concerning the primary tumor population. We demonstrated using a mouse model that tumor-associated fibroblasts are produced from the host organism and thus did not arise from transformation directly from tumor cells. We also investigated the relationship between tumor-associated fibroblasts and keratinocytes. We have shown that tumor-associated melanoma fibroblasts affect keratinocytes which, under their influence, acquire the features typically observed in migrating cells and cells undergoing epithelial-mesenchymal transition. We studied the interactions between healthy fibroblasts and tumor cells. We have demonstrated that fibroblasts acquired from healthy skin from a patient suffering from melanoma are significantly different from control fibroblasts of healthy donors in the expression profile. Changes in distal fibroblasts support the view of melanoma as a systemic disease. We have further demonstrated that melanoma-associated fibroblasts do not carry a BRAF mutation, in contrast to BRAF positivity of melanoma cells. And therefore, they did not arise from the transition from melanoma. The...
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Depletion of Treg cells for potentiation of cancer treatment with HPMA copolymer-bound cytostatic drug conjugates"
Dvořáková, Barbora ; Kovář, Marek (advisor) ; Reiniš, Milan (referee)
Tumor diseases are severe problem worldwide with increasing number of patients suffering from various types of malignancies. Many of approved therapeutics cause serious side toxicities. Therefore, there are intensive efforts to improve cancer treatment protocols. The aim of this study was to deplete regulatory T (Treg) cells without affecting other immunocompetent cells playing a positive role in tumor eradication. Treg cells were reported to hamper anti-tumor immunity and promote tumor growth and survival. Thus, their selective elimination could lead to induction of anti-tumor responses and tumor rejection if combined with chemotherapy with selected N-(2- hydroxypropyl)methacrylamide (HPMA) copolymer-bound drug conjugates. Original approach was to deplete of Treg cells without the use of anti-CD25 mAb that has been widely exploited for Treg cell elimination; however, its long-term persistence in circulation together with inhibitory effect on activated effector cells (CD25+ ) are its main disadvantages. Thus, Treg cells were sensitized to cell cycle-specific cytostatic drugs via application of IL-2/anti-IL-2 JES6.1 mAb immunocomplexes that induce vigorous selective proliferation of this cell population. Subsequent application of cell cycle-specific cytostatics showed steep decrease of Treg cell...
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Biocompatible polymer nanomaterials with non-steroid drugs tailored for anti-inflammation therapy
Běhalová, Kateřina ; Etrych, Tomáš (advisor) ; Kovář, Marek (referee)
The main aim of this bachelor thesis was synthesis and physico-chemical and preliminary biological characterization of water-soluble biocompatible polymeric system for targeted delivery of acetylsalicylic acid to inflamed tissue. Such polymer conjugates should be employed in the future for the therapeutic use within the treatment of chronic inflammatory diseases. The project included the preparation of a series of suitable acetylsalicylic acid derivatives and the synthesis of a polymeric carrier based on N-(2-hydroxypropyl) methacrylamide copolymer. Finally, the acetylsalicylic acid derivative was attached to the polymer forming a polymeric conjugate with pH-sensitive bond between the drug derivative and polymer chain, thus allowing controlled release of the drug in the target site. The work includes SEC, NMR and HPLC characterization of all synthesized compounds, verification of their stability in solutions with different pH and results of the release of drug derivatives from the polymeric carrier at physiological pH, or mildly acidic pH modeling inflamed environment. Moreover, the cytotoxicity bioassay of acetylsalicylic acid derivatives using LDH assay did not prove any cytoxicity at the level of necrotic activity. Also, their cyclooxygenase 1 inhibitory activity was observed and discussed. The polymeric...
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Intercellular interactions in skin tumors.
Kučera, Jan ; Smetana, Karel (advisor) ; Masařík, Michal (referee) ; Kovář, Marek (referee)
The dissertation is focused on the study of intercellular interactions in skin tumors. It is based on 5 original publications that cover several topics. We studied the origin of tumor-associated fibroblasts concerning the primary tumor population. We demonstrated using a mouse model that tumor-associated fibroblasts are produced from the host organism and thus did not arise from transformation directly from tumor cells. We also investigated the relationship between tumor-associated fibroblasts and keratinocytes. We have shown that tumor-associated melanoma fibroblasts affect keratinocytes which, under their influence, acquire the features typically observed in migrating cells and cells undergoing epithelial-mesenchymal transition. We studied the interactions between healthy fibroblasts and tumor cells. We have demonstrated that fibroblasts acquired from healthy skin from a patient suffering from melanoma are significantly different from control fibroblasts of healthy donors in the expression profile. Changes in distal fibroblasts support the view of melanoma as a systemic disease. We have further demonstrated that melanoma-associated fibroblasts do not carry a BRAF mutation, in contrast to BRAF positivity of melanoma cells. And therefore, they did not arise from the transition from melanoma. The...
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Overcoming cancer resistance to chemotherapy through HPMA copolymer conjugates
Sivák, Ladislav ; Kovář, Marek (advisor) ; Smetana, Karel (referee) ; Palich Fučíková, Jitka (referee)
Multidrug resistance (MDR) is a common cause of failure in chemotherapy for malignant diseases. Cancer cells develop MDR most often via the up-regulation of P- glycoprotein (P-gp) expression. P-gp is an efflux pump with broad specificity belonging to ATP-binding cassette (ABC) transporters which decreases the intracellular concentration of various drugs. We designed polymeric conjugates based on an N-(2-hydroxypropyl)methacrylamide (HPMA) bearing a cytostatic drug and/or P-gp inhibitor and tested their cytostatic/cytotoxic activity in vitro and their therapeutic efficacy in vivo in MDR tumors. We demonstrated that HPMA copolymer conjugates bearing both the cytostatic drug (doxorubicin (Dox) or pirarubicin) and the P-gp inhibitor (derivative of reversin 121 (R121) or ritonavir) possess remarkable cytostatic and cytotoxic activity in MDR tumor cell lines in vitro and superior antitumor activity in vivo. Notably, the HPMA copolymer conjugate bearing both Dox and R121 showed significant antitumor activity in both P388/MDR and CT26 mouse tumor models and was capable to completely cure 6 out of 8 mice with established CT26 tumors. We explored the potential of micelle-forming HPMA copolymer-poly(propylene oxide) (PPO) diblock bearing Dox to overcome MDR in vitro and in vivo. The HPMA copolymer-PPO diblock...
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Antitumor activity of IL-2 and IL-7 immunocomplexes in combination with αCTLA-4 and αPD-1 mAbs
Hnízdilová, Tereza ; Kovář, Marek (advisor) ; Hájková, Michaela (referee)
Biological activity of IL-2 and IL-7 in vivo is significantly increased when complexed with some of the respective anti-cytokine mAb. Different immune cell subsets can be preferentially stimulated depending on the anti-IL-2 mAb used to complex IL-2. IL-2/anti-IL-2 mAb S4B6 immunocomplexes (IL-2/S4B6) induce preferential expansion of CD122high cells whereas IL-2/anti-IL-2 mAb JES6-1 immunocomplexes (IL-2/JES6-1) highly selectively stimulate CD25high cells in mice. Similarly, IL-7/anti-IL-7 mAb M25 immunocomplexes (IL-7/M25) possess higher stimulatory activity for both naïve and memory CD8+ T cells in vivo in comparison to free IL-7. CTLA-4 and PD-1 molecules are inhibitory receptors which negatively regulate proliferation, survival and effector functions of T cells. Blocking antibodies against these molecules represent promising immunotherapeutic tool for treatment of malignant diseases. We examined possible synergism of IL-2/S4B6 and αCTLA-4 plus αPD-1 mAbs in tumor-bearing mice. We found that the expansion of recently activated CD8+ T cells driven by IL-2/S4B6 was further augmented by αCTLA-4 plus αPD-1 mAbs. However, these two immunotherapeutic approaches did not show synergistic antitumor activity in any mouse tumor model tested. Next, we showed that IL-7/M25 possessed higher biological activity...
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Advances in chemotherapy and novel antitumor drugs
Kraus, Michal ; Kovář, Marek (advisor) ; Koudelková, Lenka (referee)
Cancer is among the leading causes of death worldwide. While some types of cancer became almost entirely curable, majority of malignant tumors are still potentially deadly diseases due to unsensitivity of tumors to conventional chemotherapy or diversity of cancer cells within the tumor and subsequent development of resistance. The underlying mechanism of action of conventional antitumor drugs is mostly related to cell division. DNA damage, inhibition of DNA synthesis and repair or disrupted formation of mitotic spindle are the most common mechanisms. However, it implies that most of the drugs are cytotoxic for rapidly dividing cells in general which results in variety of undesirable side effects for patients. Search for novel anticancer drugs targeting cancer cells more selectively has been point of interest of researchers for decades. Hundreds of new potential anticancer drugs are being described every year, some posessing so far unrecognized mechanisms of action. Process called drug repurposing examines drugs that have already been approved for clinical use in other than oncology field and results into discovering of interesting "novel" anticancer agents. Another general trend is represented by shift towards development of targeted therapy which is slowly replacing traditional cytotoxic...
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The role of galectins in cancer cell invasiveness
Remišová, Michaela ; Brábek, Jan (advisor) ; Kovář, Marek (referee)
Galectins are family of β-galactosidase binding proteins that serve many functions in all kind of mammalian cells. In the past years galectins, namely galectin-1 and galectin-3, have been revealed to play a major role in various cancer processes including cancer cell invasiveness, a process indispensable for the formation of metastasis. Both extracellular and intracellular forms of galectins modify the process of invasiveness in various ways, through interacting with different components of the cell or of the cell signalling pathways. The aim of this bachelor's thesis is to summarize mechanisms by which galectins promote cancer cell invasiveness. Keywords: galectins, galectin-1, galectin-3, invasiveness, cancer, metastasis
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Anti-tumor activity and toxicity of HPMA copolymer conjugates bearing cytostatic drug
Tomalová, Barbora ; Kovář, Marek (advisor) ; Smetana, Karel (referee) ; Špíšek, Radek (referee)
7 ABSTRACT In this study, we addressed the biological activity and pharmacological features of selected HPMA copolymer-based drug conjugates. We determined their cytostatic activity in vitro as well as toxicity in vivo and therapeutic effcicacy in mouse tumor models. Assessment of maximum tolerated dose (MTD) of two structurally different HPMA copolymer-based conjugates bearing doxorubicin (DOX) attached via pH-sensitive hydrazon bond (HPMA- DOXHYD ) showed that high molecular weight non-degradable star HPMA-DOXHYD conjugate possesses relatively low MTD ~22.5 mg DOX/kg, while linear HPMA-DOXHYD has MTD ~85 mg DOX/kg. Thus, MTD of linear conjugate is 3.7 times higher than that of the star conjugate. Subsequently, we reported that linear conjugate proved to be more efficient in case of treatment of solid tumor EL4 lymphoma and star conjugate to be superior in case of BCL1 leukemia treatment. We also compared biological activity of star and linear HPMA copolymer-based conjugates bearing docetaxel (DTX) attached via pH-sensitive hydrazon bond (HPMA-DTXHYD ). MTD of star conjugate (~160 mg DTX/kg) was proved to be 4 times higher than MTD od free DTX (40 mg/kg). We were not able to determine MTD of linear conjugate as it exceeded 200 mg DTX/kg (the highest soluble dose we were able to administer as a bolus)....
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