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Povzetek: In genetic toxicology, there is a trend against the increased use of in vivo models as highlighted by the 3R strategy, thus encouraging the development and implementation of alternative models. Two-dimensional (2D) hepatic cell models, which are generally used for studying the adverse effects of chemicals and consumer products, are prone to giving misleading results. On the other hand, newly developed hepatic three-dimensional (3D) cell models provide an attractive alternative, which, due to improved cell interactions and a higher level of liver-specific functions, including metabolic enzymes, reflect in vivo conditions more accurately. We developed an in vitro 3D cell model from the human hepatocellular carcinoma (HepG2) cell line. The spheroids were cultured under static conditions and characterised by monitoring their growth, morphology, and cell viability during the time of cultivation. A time-dependent suppression of cell division was observed. Cell cycle analysis showed time-dependent accumulation of cells in the G0/G1 phase. Moreover, time-dependent downregulation of proliferation markers was shown at the mRNA level. Genes encoding hepatic markers, metabolic phase I/II enzymes, were time-dependently deregulated compared to monolayers. New knowledge on the characteristics of the 3D cell model is of great importance for its further development and application in the safety assessment of chemicals, food products, and complex mixtures.
Objavljeno v DiRROS: 22.07.2024; Ogledov: 169; Prenosov: 147
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Povzetek: Glioblastoma (GB), is the most common and aggressive malignant primary brain tumour in adults. Intra- and inter-tumour heterogeneity, infiltrative GB cell invasion and presence of therapy-resistant GB stem cells (GSCs) represent major obstacles to favourable prognosis and poor therapy response. Identifying the biomarkers of the most aggressive tumour cells and their more efficient targeting strategies are; therefore, crucial. Recently, transcription factor TRIM28 has been identified as a GB biomarker and, in this study, we have shown high expression of TRIM28 in GB and in low grade gliomas as well as higher expression in GSCs vs. differentiated GB cells, although in both cases not significant. We demonstrated significant in vitro inhibition of GB cells and GSCs invasiveness and spread in zebrafish brains in vivo by anti-TRIM28 selective nanobody NB237. TRIM28 was also enriched in GB (tumour) core and associated with the expression of stem cell genes, but was not prognostic for overall survival. However, based on the above results, we conclude that TRIM28 nanobody NB237 offers a new opportunity as a GB therapeutic tool.
Objavljeno v DiRROS: 19.07.2024; Ogledov: 148; Prenosov: 148
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Povzetek: The major weakness of the current in vitro genotoxicity test systems is the inability of the indicator cells to express metabolic enzymes needed for the activation and detoxification of genotoxic compounds, which consequently can lead to misleading results. Thus, there is a significant emphasis on developing hepatic cell models, including advanced in vitro three-dimensional (3D) cell-based systems, which better imitate in vivo cell behaviour and offer more accurate and predictive data for human exposures. In this study, we developed an approach for genotoxicity testing with 21-day old spheroids formed from human hepatocellular carcinoma cells (HepG2/C3A) using the dynamic clinostat bioreactor system (CelVivo BAM/bioreactor) under controlled conditions. The spheroids were exposed to indirect-acting genotoxic compounds, polycyclic aromatic hydrocarbon [PAH; benzo(a) pyrene B(a)P], and heterocyclic aromatic amine [PhIP]) at non-cytotoxic concentrations for 24 and 96 h. The results showed that both environmental pollutants B(a)P and PhIP significantly increased the level of DNA strand breaks assessed by the comet assay. Further, the mRNA level of selected genes encoding metabolic enzymes from phase I and II, and DNA damage responsive genes was determined (qPCR). The 21-day old spheroids showed higher basal expression of genes encoding metabolic enzymes compared to monolayer culture. In spheroids, B(a)P or PhIP induced compound-specific up-regulation of genes implicated in their metabolism, and deregulation of genes implicated in DNA damage and immediate-early response. The study demonstrated that this model utilizing HepG2/C3A spheroids grown under dynamic clinostat conditions represents a very sensitive and promising in vitro model for genotoxicity and environmental studies and can thus significantly contribute to a more reliable assessment of genotoxic activities of pure chemicals, and complex environmental samples even at very low for environmental exposure relevant concentrations.
Ključne besede: in vitro 3D cell model, 21-day old spheroids, cytotoxic, genotoxic, gene expression
Objavljeno v DiRROS: 19.07.2024; Ogledov: 144; Prenosov: 168
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Povzetek: Glioblastomas remain the most lethal primary brain tumors. Natural killer (NK) cell-based therapy is a promising immunotherapeutic strategy in the treatment of glioblastomas, since these cells can select and lyse therapy-resistant glioblastoma stem-like cells (GSLCs). Immunotherapy with super-charged NK cells has a potential as antitumor approach since we found their efficiency to kill patient-derived GSLCs in 2D and 3D models, potentially reversing the immunosuppression also seen in the patients. In addition to their potent cytotoxicity, NK cells secrete IFN-γ, upregulate GSLC surface expression of CD54 and MHC class I and increase sensitivity of GSLCs to chemotherapeutic drugs. Moreover, NK cell localization in peri-vascular regions in glioblastoma tissues and their close contact with GSLCs in tumorospheres suggests their ability to infiltrate glioblastoma tumors and target GSLCs. Due to GSLC heterogeneity and plasticity in regards to their stage of differentiation personalized immunotherapeutic strategies should be designed to effectively target glioblastomas.
Ključne besede: glioblastoma, natural killer cells, translational oncology
Objavljeno v DiRROS: 16.07.2024; Ogledov: 224; Prenosov: 203
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Povzetek: Tyrosine kinase inhibitors (TKIs) are designed for targeted cancer therapy. The consumption of these drugs during the last 20 years has been constantly rising. In the zebrafish (Danio rerio) embryo toxicity test, we assessed the toxicity of six TKIs: imatinib mesylate, erlotinib, nilotinib, dasatinib, sorafenib and regorafenib. Imatinib mesylate and dasatinib induced lethal effects, while regorafenib, sorfenib and dasatinib caused a significant increase of sub-lethal effects, predominantly oedema, no blood circulation and formation of blood aggregates. The analyses of the changes in the expression of selected genes associated with the hormone system after the exposure to imatinib mesylate, dasatinib and regorafenib demonstrated that all three tested TKIs deregulated the expression of oestrogen receptor esr1, cytochrome P450 aromatase (cypa19b) and hydroxysteroid-dehydrogenase (hsd3b), regorafenib, and also thyroglobulin (tg). The expression of genes involved in the DNA damage response (gadd45 and mcm6) and apoptosis (bcl2) was deregulated only by exposure to regorafenib. The data indicate that common mechanisms, namely antiangiogenic activity and interference with steroidogenesis are involved in the TKI induced sub-lethal effects and potential hormone disrupting activity, respectively. The residues of TKIs may represent an environmental hazard; therefore, further ecotoxicological studies focusing also on the effects of their mixtures are warranted.
Ključne besede: aquatic toxicity, tyrosine kinase inhibitors, zebrafish embryo toxicity test, gene expression, environmental hazard
Objavljeno v DiRROS: 16.07.2024; Ogledov: 183; Prenosov: 122
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