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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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Activation and function of BH3-only proteins from the Bcl-2 family
Peterka, Martin ; Anděra, Ladislav (advisor) ; Pavlů, Barbora (referee)
The Bcl-2 (B-cell lymphoma-2) family of proteins comprises crucial regulators of apoptosis in metazoans. In mammals, Bcl-2 family members regulate mitochondrial outer membrane permeabilization (MOMP), which leads to the release of pro-apoptotic molecules from the mitochondrial intermembrane space and subsequent apoptosis. Defects in the Bcl-2 signaling can result in various pathological conditions including cancer. BH3-only proteins form a pro-apoptotic subset of the Bcl-2 family. They mainly act as sensors of various pro-apoptotic stimuli such as cytokine deprivation, genotoxic stress, activated "death receptors" and others. In mammals, over ten BH3-only proteins have been identified, eight of which have been linked to the regulation of MOMP. These "conventional" BH3-only proteins are Bim, Bid, Puma, Noxa, Bad, Hrk, Bik and Bmf. This thesis provides an overview of their origin, structure, activation and involvement in apoptosis.
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The role of erbB-2 oncogene in the biology of cancer stem-like cells
Prokopová, Kateřina ; Neužil, Jiří (advisor) ; Anděra, Ladislav (referee)
Recent studies indicate the existence of a subpopulation of cells within tumours with stem cell-like characteristics. These "cancer stem-like cells" (CSCs) are relatively resistant to established therapies, usually targeting differentiated and fast proliferating cells. Therefore, CSCs may be a reason for the relapse of neoplastic diseases. CSCs can be characterised by a specific gene expression profile and deregulated signalling pathways. Of these, upregulation of the erbB-2 (HER2) receptor, a hallmark of ~25-30% breast cancer patients, is related to dismal prognosis, elevated proliferation potential and resistance to chemotherapy. Recent evidence has suggested that upregulation of erbB-2 leads to increase in the pool of CSCs. In our study we used mammospheres, cells grown in the absence of serum, an in vitro model of breast CSCs, which were prepared by "weaning" breast cancer MCF7 cells to a special medium. These cells were CD44high and showed increased expression of ABCG-2, Sox-2, Vimentin as well as high levels of erbB-2. Next, we prepared a stable line of MCF7 cells with low levels of erbB-2 by shRNA. ErbB-2low cells were characterised for expression of set of CSCs markers and tested for tumour forming efficacy in nude mice using ultrasound imaging. Keywords Cancer stem-like cells, erbB-2,...
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Posttranslational modification of the adapter protein DAXX in the cellular response to genotoxic stress
Bražina, Jan ; Anděra, Ladislav (advisor) ; Černý, Jan (referee) ; Vodička, Pavel (referee)
Maintaining the chromosome continuity and complete genetic information in human cells is crucial for cell survival and the whole organism. It prevents life-threatening pathologies and preserves genetic continuity. However, cellular DNA is exposed to both endogenous and exogenous stress damaging its content and integrity. This stress activates mechanisms involving detection and repair of these damaged sites (DDR). One of the most serious types of DNA damage double-stranded breaks (DSB) occuring when both strands are severed. DSBs trigger wave of PTMs that regulate protein interactions, nuclear localization and catalytic activity of hundreds of proteins. Such modifications include acetylation, methylation, SUMOylation, ubiquitinylation and especially phosphorylation. The most important kinases involved in DDR kinases are ATM, ATR and DNA-PK. These kinases are activated immediately after the detection of the damaged area. DAXX (Death-associated protein 6) is an adapter and predominantly nuclear protein, which is involved in chromatin remodeling, gene expression modulation, antiviral response and depositing histone H3.3 variants into chromatin or telomeres. Daxx is essential for murine embryogenesis, since the homozygous deletion is lethal in E9.5-10. In 2006 a study mapping the substrates of kinases...
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