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Inhibition of P-glycoprotein-mediated multidrug resistance and STAT3 signaling pathway through polymeric conjugates bearing protease inhibitor derivatives
Starenko, Daniil ; Kovář, Marek (advisor) ; Truksa, Jaroslav (referee)
Tumor cells expressing high levels of some ABC transporters (mainly P-glycoprotein) can become resistant to many structurally and functionally different drugs. Such multidrug resistance can be a significant barrier for a successful chemotherapy of malignant diseases. There is a considerable amount of small-molecular-weight compounds capable of potent inhibition of P-glycoprotein, but none of them are approved for the clinical use. STAT3 is a transcription factor important for many physiological processes, but its constitutive activation may lead to the malignant transformation and chemotherapy resistance in tumor cells. This molecule is thus potential target for anticancer drugs. The inhibition of STAT3 signaling should lead to lower cancer cell proliferation and their increased susceptibility to induction of apoptosis. Considerable attention is given to increase the effectiveness and to lower the adverse effects of conventional cytostatic agents via using nanomaterials and drug delivery systems in the research of new cancer therapy approaches. Polymeric carriers based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers are promising candidates in this field. The main aim of this diploma thesis was to evaluate the effectiveness of several HIV protease inhibitor (ritonavir, lopinavir, indinavir,...
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The role of γc cytokines in the immune system and cancer immunotherapy
Ptáček, Bohumil ; Kovář, Marek (advisor) ; Adkins, Irena (referee)
Cytokines are proteins produced mostly by cells of hematopoietic origin and transduce signals via engaging cell surface receptors on either the cytokine-producing cells (autocrine signaling) or other target cells (paracrine signaling). Common cytokine receptor subunit (γc) cytokines are small glycoproteins belonging to type I cytokines with pleiotropic activities in both the innate and adaptive immune systems. All γc cytokines share a γc receptor subunit in their complete receptors. The first part of this thesis aims to summarize information about the biology of γc cytokines, their receptors, and their role in the immune system and its functions. The second part discusses the use of γc cytokines in cancer immunotherapy, presenting examples of particular γc cytokine therapies, and describes the approaches to improve the pharmacological features of γc cytokines or efficiently combine them with other immunotherapies and anticancer treatments. Keywords: γc cytokine, cytokine receptor, T cell, NK cell, cancer immunotherapy
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Antitumor activity of the polymeric conjugates bearing derivatives of protease inhibitors and conventional cytostatics for the treatment of head and neck tumors
Běhalová, Kateřina ; Kovář, Marek (advisor) ; Vodička, Pavel (referee)
Head and neck cancers account for about 4,6 % of all malignancies worldwide and their incidence is increasing. However, the development of chemotherapeutics in this field is rather stagnating. One promising approach seems to be the repurposing of drugs originally developed and clinically used as HIV protease inhibitors, which have also been described to have anticancer activity. Esterification of the OH group of these drugs with 5-methyl-4-oxohexanoic acid allows their attachment via pH-sensitive hydrazone linkage to a hydrophilic and biocompatible HPMA copolymer carrier. This binding provides an improvement in the pharmacokinetics of the drug, prolongs its circulation time in the bloodstream, lowers its side effects and it also allows passive accumulation of the drug in the tumor tissue due to the EPR effect. Six protease inhibitors in total (ritonavir, lopinavir, saquinavir, indinavir, nelfinavir and atazanavir) were derivatized as described above. Ritonavir and its derivative had been tested in a previous project and were used as reference substances. After initial determination of in vitro cytostatic and cytotoxic activity in FaDu (human head and neck carcinoma), SCC7 (murine squamous cell carcinoma), 4T1 (murine breast carcinoma) and CT26 (murine colon carcinoma) cell lines, lopinavir derivative was...
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Polymer probes for photodynamic therapy of solid tumors
Kotalík, Kevin ; Etrych, Tomáš (advisor) ; Kovář, Marek (referee)
One of the currently studied promising strategies in advanced oncologic treatment is photodynamic therapy, a method based on the administration of so-called photosensitisers, i.e. photoactive compounds such as porphyrins, and subsequent irradiation of tumor tissue with light of appropriate wavelength. An excitation of the photosensitiser, present in the tumor area, is hence invoked and reactive oxygen species (ROS) are formed. These species afterward cause the apoptosis of the tumor cells, leading to destruction of tumor tissue. In photodynamic therapy, the strategy of administration of a prodrug which is metabolised to the active photosensitiser can be used with advantages. In photodynamic therapy, this prodrug may be 5-aminolevulinic acid (5-ALA) or its esters which are metabolised to protoporphyrin IX (PPIX), the photosensitiser proper. The targeted drug delivery to the tumor tissue can be achieved by using various delivery systems, e.g. water-soluble polymer conjugates carrying the drug. Due to their size, these polymer conjugates are accumulated in solid tumors on the basis of the enhanced permeability and retention (EPR) effect. Macromolecules can penetrate the tumor vasculature, which, unlike that of healthy tissue, is imperfectly developed and contains gaps between endothelial cells....
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Interaction of mesenchymal stem cells with immune system and their use in cancer therapy
Sivák, Ladislav ; Kovář, Marek (advisor) ; Paňková, Daniela (referee)
Mesenchymal stem cells (MSC) are multipotent progenitor cells with the ability to differentiate into ectodermal, mesodermal and endodermal cell line. They interact with innate and adaptive immunity and modulate their effector functions. Immunoregulatory effect of MSC results in suppression of inflammatory immune response and induces anti-inflammatory immune response. In addition, MSC have the ability to migrate into tumor site trough soluble factors produced by tumor cells and contribute to tumor growth and metastasis. Preferential homing to site of cancer growth and regulation of immune system make the MSC a promising tool for cancer therapy. Key words: mesenchymal stem cells, immunoregulation, tumors, cancer gene therapy
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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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Cancer treatment with polymer-bound cytostatic drugs and its potentiation through immunomodulation
Chmelová, Helena ; Kovář, Marek (advisor) ; Drda Morávková, Alena (referee)
Cancer treatment with polymer-bound cytostatic drugs and its potentiation through immunomodulation Poly[N-2-(hydroxypropyl)-methacrylamide] (PHPMA) is a synthetic water soluble and biocompatible polymer which can be used as a carrier of a cytostatic drug and an antibody as a targeting moiety. The antibody ensures the site-specific delivery of the conjugate. Nevertheless, even polymeric conjugates without any tumor-specific targeting moiety are passively accumulated within solid tumors via so called Enhanced Permeability and Retention (EPR) effect, in case that their molecular wight is at least 40 kDa. Antibody-targeted polymeric drugs have been shown previously to have a cytostatic activity in vitro and an antitumor activity in vivo. Since treatment of cancer diseases in practice is far from such ideal conditions and many tumors have no strictly specific marker suitable for targeted therapy, upgrading of the treatment efficacy represents the major challenge. One of the possible ways how to improve insufficient chemotherapy outcome can be using of a combination of polymer-bound cytostatic drug and potent immunomodulation able to induce a robust anti-cancer immune response. In this study, we have used B cell leukemia BCL1 as an experimental tumor model. BCL1 cells express surface IgM with an unique...
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Molecular and functional characterization of the death receptor 6
Klíma, Martin ; Anděra, Ladislav (advisor) ; Živný, Jan (referee) ; Kovář, Marek (referee)
Death receptor-6 (DR6/TNFRsf21/CD358) is a receptor from the TNFR superfamily that likely participates in the regulation of proliferation and differentiation of T- and B-lymphocytes and neural cells. The 655-amino acid human DR6 is a type I transmembrane protein containing four cysteine-rich domains in its extracelular part and a death domain followed by the CARD-like region in its cytoplasmic part. Overexpression of DR6 in some cell lines leads to apoptosis, and/or to activation of nuclear factor NF-κB and stress kinases of the JNK family. In the first part of our work we focused on molecular characterization of DR6, including the analysis of its posttranslational modifications. We found that DR6 is an extensively posttranslationally modified protein including S-palmitoylation and both N- and O-glycosylation. Six N-glycosylation and one S-palmitoylation sites were precisely mapped to appropriate asparagines and cysteine respectively. The juxtaposed linker region (between cystein-rich domains and the transmembrane part), which also contains Ser/Thr/Pro-rich region with clustered putative O-glycosylation sites, is required for the plasma membrane localization of DR6. N-glycosylation, but interestingly not S-palmitoylation, may play a role in targeting of DR6 into detergent-resistant...
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Tumor microenvironment and the importance of anti-tumor immunity for clinical course of human cancers
Partlová, Simona ; Špíšek, Radek (advisor) ; Drbal, Karel (referee) ; Kovář, Marek (referee)
Cancer development and progression vary depending on tumor type, localization, invasion, immunogenicity and the ability of immune system to become activated. There are frequent interactions between tumor cells and immune cells, occuring locally at the site of primary tumor or distally through paracrine signalling of various mediators and cytokines. The main subject of this PhD thesis is to study key factors and aspects of immune response in cancer patients. In the first part, we analyzed immune cells infiltrating tumor tissues of ovarian cancer patients at different stages of disease. We focused on the dynamics of immune response, primarily on frequency of individual T lymphocyte populations in peripheral blood and tumor infiltrating T lymphocytes in tumors of early and advanced stages of ovarian cancer. We found that during disease progression there is a gradual decrease of proinflammatory Th17 and Th1 immune responses and a specific recruitment of regulatory T cells to the tumor site, which results in a significant immune suppression in the tumor microenvironment. In the second part, we demonstrated that the character of immune response in HPV-positive head and neck cancer patients is very different from the patients with tumors not associated with HPV infection. In HPV-positive patients, significantly...
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