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Role of signaling pathways and redox processes in cancer cell biology
Vališ, Karel ; Kovář, Jan (advisor) ; Anděra, Ladislav (referee) ; Kozubík, Alois (referee)
5 identifikovali VDAC2 protein jako možný transportér železa do nádorových buněk a tudíž možný cíl protinádorové terapie. Abstract Specific apoptosis induction in cancer cells represents promising way of anticancer therapy without damaging healthy tissues. Hence, search for new molecular targets capable of specific apoptosis triggering is highly challenging, mainly due to new and effective anti-cancer therapies development. Cancer cell metabolism (1) and mainly mitochondrial metabolism (2) seems to represent intriguing target. Mitochondria play essential role in life of the cell but on the other hand mitochondria activate cell death which is usually connected to increased ROS (reactive oxygen species) production. Contribution of this work is identification of vitamin E analogues as inhibitors of complex II and potent inductors of mitochondrial ROS in cancer cells. Additionally, we have demonstrated activation of conserved Hippo/Mst1 kinase in response to the vitamin E analogues which results in FoxO1 nuclear localization, NOXA gene transactivation, Bak protein activation, mitochondrial membrane permeabilisation and apoptosis induction. Next we searched for membrane proteins with increased expression in cancer cells exposed to iron deprivation. These proteins can play a role in iron metabolism of cancer...
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Functional assessment of Bcl-2 family proteins in mitochondrial metabolism and beyond
Sovilj, Dana ; Anděra, Ladislav (advisor) ; Mráček, Tomáš (referee) ; Živný, Jan (referee)
(CZ) Od jejich prvotní identifikace v háďátku C. elegans a také v lidských buňkách před více než 30 lety jsou proteiny z rodiny Bcl-2 spojovány s indukcí, regulací a potlačením mitochondriální apoptotické signalizace, ale také se mohou uplatňovat při modulaci neapoptotických signálních dráh. V této studii jsme si stanovili za hlavní cíl rozšířit stávající znalosti o neapoptotických rolích hlavních proapoptotických proteinů z Bcl-2 rodiny, BAX a BAK, zejména pak na jejich roli v buněčném metabolismu. Pomocí genové editace využitím CRISPR/Cas9 jsme eliminovali expresi těchto proteinů v lidských nádorových buňkách různého tkáňového původu a v těchto Bax/Bak- deficitních buňkách jsme primárně analyzovali mitochondriální respiraci a buněčnou glykolýzu. Zatímco eliminace exprese Bax a Bak neměla žádný patrný vliv na glykolýzu ve všech testovaných buněčných liniích, v závislosti na buněčném typu modulovala mitochondriální respiraci. Eliminace exprese Bax a Bak v buňkách rakoviny tlustého střeva HCT-116 neměla vliv na mitochondriální respiraci, ale zjevně ovlivnila mitochondriální respiraci v Bax/Bak-deficitních buňkách odvozených od glioblastomu (U87) a lymfomů (HBL-2, UPF1H, UPF1G). Bax/Bak -/- buňky U87 významně upregulovaly mitochondriální respiraci a akcelerovaly svou proliferaci a také nádorový růst...
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Antitumor activity of HPMA copolymer-bound gemcitabine conjugate potentiation by IAP inhibitors
Ivančík, Martin ; Kovář, Marek (advisor) ; Anděra, Ladislav (referee)
Cancer represents a significant global health challenge, with a substantially increasing incidence. There is a critical need for enhanced prevention, early detection, and improved therapeutic approaches to treat malignant diseases. While chemotherapy remains a standard treatment for many cancers, its efficacy is often limited by cytotoxic effects on healthy cells and numerous side toxicities. In response to these challenges, novel strategies such as combination therapies and drug delivery systems have emerged. In this project, we investigated the cytostatic and cytotoxic effects of two inhibitors of IAP (inhibitor of apoptosis) proteins, LCL-161 and AZD5582, and their potential to boost the anticancer activity of gemcitabine. These compounds were evaluated in five human cancer cell lines: pancreatic (PANC1, BxPC- 3, MiaPaca-2), prostatic (PC-3), and breast (MDA-MB-231) carcinomas. Both IAP inhibitors demonstrated anticancer activity as single agents, with the MiaPaca-2 and BxPC-3 cell lines showing the highest sensitivity. No correlation was observed between the expression levels of IAP genes (cIAP1, cIAP2, XIAP) and the sensitivity of cell lines to IAP inhibitors. However, four of five tested cell lines possessed a consistent pattern in the expression of these three genes. IAP inhibitors were able...
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Characterization and modulation of MitoTam-induced cell death in breast carcinoma cells
Hrysiuk, Mariia ; Anděra, Ladislav (advisor) ; Dráber, Peter (referee)
Although recent years brought many breakthrough discoveries in anti-cancer research and therapy, malignant diseases such as breast cancer (BC) still present one of the major health threats worldwide. Cancer cells usually gain resistance to the activation of regulated cell death (RCD) modalities such as caspase-dependent apoptosis. Among novel RCD-inducing agents belongs to mitochondria-targeted tamoxifen - MitoTam, which is also the major focus of this Thesis. In a panel of BC cells, we determined the energetic (mitochondrial respiration vs. glycolysis) and major RCD-related proteins (Western blotting) profiles, and using Lumascope LS720-assisted time-lapse monitoring we analyzed their sensitivity to MitoTam-induced RCD. We found out that glycolysis-preferring BC cells as MDA-MB-231 are more resistant to MitoTam treatment than mitochondrial respiration-biased MDA-MB-453 cells. However,the majority of tested BC cells can be sensitized to MitoTam by BH3 mimetics such as BCL-XL targeting A1155463 and some cellular metabolism-modulating compounds such as lactate dehydrogenase inhibitor (R)-GNE-140, especially in the pre-treatment regime. Also, other metabolism-modulating compounds such as Pyruvate Dehydrogenase Kinases inhibitor JX06 potently enhanced the efficacy and kinetics of MitoTam-induced RCD....
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The role of caspase-3 in apoptosis
Kolářová, Karolína ; Tlapáková, Tereza (advisor) ; Anděra, Ladislav (referee)
Caspases provide anti-inflammatory, apoptotic and developmental processes in organisms. They are enzymes with a wide range of activities in all cells, and various pathogeneses can occur if their proper function is disturbed. Since the 1990s, caspases have been a topic of interest for scientists, as their direct link to the triggering of apoptotic processes is a promising possibility for the therapy of diseases related to apoptosis, such as cancer, neurodegenerative diseases, but also cardiac ischemia and diabetes. The cascade of apoptotic processes is controlled by the aforementioned caspases, which are located in the caspase cascade. When the cascade is triggered in a cell, it is due to the presence of a "danger" signal, which can be very different. The most well-known triggers of the apoptotic cascade include activated Fas receptor and FasL ligand, cytochrome c present in the cytoplasm, an imbalance of IAPs in the cell, damaged DNA, and many others. Upon receipt of a signal, initiator caspase-2, caspase-8, and caspase-9 are activated, which in turn activate effector caspases-3, caspase-6, and caspase- 7, cleaving many substrates to promote apoptosis. Thus, caspase-3 is the effector enzyme responsible for the actual execution of apoptosis. However, caspase-3 properties are not only apoptotic, it...
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Role of DD- and DED-containing adaptor proteins in apoptotic signaling
Čaja, Fabián ; Anděra, Ladislav (advisor) ; Janštová, Vanda (referee)
Proteins containing a bundle of six anti-paralel α-helices in so-called "death domain" (DD) and similar structures (DED, CARD) represent important players in apoptotic signaling. To DD/DED/CARD domains-containing proteins belong pro- apoptotic membrane receptors from the TNFR superfamily, then adaptor proteins and enzymes as proteases or kinases. These pro-apoptotic "death receptors" interact with adaptor proteins and initiator caspases containing DDs or DEDs and activate apoptotic signaling cascade. DEDs and DDs are in addition found in many proteins participating in activation of caspases or other non-apoptotic signaling. Many experimental models document that defects in and deregulations of proteins containing DDs and DEDs can have severe if not lethal consequences for an organism. Abberations in these proteins in many cases could lead to cancerogenesis, immunodeficiencies or developmental defects.
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Effect of cancer-associated fibroblasts on the survival, proliferation and invasiveness of cancer cells.
Nováková, Gita ; Anděra, Ladislav (advisor) ; Brábek, Jan (referee)
Tumour microenvironment, in addition to cancer cells themselves, represents important structural and functional part of the tumour. Similarly to the normal organs tumour microenvironment comprises several cell types (fibroblasts, immune cells, endothelial cells etc.) and non-cellular components, particularly extracellular matrix. All of them form favourable conditions for the growth, proliferation, protection from the immune system- mediated destruction and nutrition of cancer cells. Cancer associated fibroblasts (CAFs) represent the most abundant cell type of tumour microenvironment. Their origin can be traced to local normal fibroblasts, endothelial cells or epithelial cells and the transition into the CAFs phenotype is influenced with several factors secreted by cancer cells (particularly TGF-β). In contrast to fibroblasts activated during wound healing newly formed cancer associated fibroblasts expressing α-SMA are not subsequently eliminated from the respektive tissue. They persist and produce a number of pro-tumorigenic factors - SDF-1, HGF, IGF-1, IL-6, VEGF, PDGF-C, TGF-β, MMPs etc. CAFs and their secreted factors target several signalling pathways enhancing basic characteristics of the tumour, so called Hallmarks of Cancer. Cancer associated fibroblasts promote proliferation and invasiveness of...
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Immunogenic cancer cell death triggered by free and polymer bound doxorubicin
Kabešová, Martina ; Kovář, Lubomír (advisor) ; Anděra, Ladislav (referee)
Immunogenic cancer cell death triggered by free and polymer-bound doxorubicin Water-soluble polymeric drug carriers based on N-(2-hydroxypropyl) methacrylamide (HPMA) have been developed to avoid undesirable side-effects of systemically active cytostatic drugs. Conjugates based on HPMA copolymer passively accumulate in tumor tissue of solid tumors due to the effect of enhanced vascular permeability and retention effect (EPR effect). Active accumulation can be achieved by binding of targeting structure recognising tumor-specific receptors to the polymeric carrier. Conjugation of free doxorubicin to HPMA polymeric carrier significantly reduces its nonspecific side effects in vivo by maintaining effective antitumor activity in vitro and in vivo. Besides direct cytotoxic effect on tumor cells, HPMA conjugates with bound doxorubicin possess immunostimulatory properties and induce long-lasting anti-tumor immunity in cured mice. Recent studies have shown that free doxorubicin induce immunogenic apoptosis in tumor cells. Those cells are dying by cell death which possesses characteristic markers of apoptosis but these cells are able to activate the immune system and thus induce effective anti-tumor immune response. This phenomenon has been described as crucial for a development of treatment-induced...
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