There is a significant emphasis to develop hepatic cell models, including advanced in vitro three-dimensional (3D) cell-based systems, which better imitate in vivo cell behavior 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. The results showed that both environmental pollutants 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. 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.
COBISS.SI-ID: 36593667
In the study, we used the advanced 3D cell model developed from human hepatocellular carcinoma (HepG2) cells for the evaluation of cylindrospermopsin cyto-/genotoxic activity. Spheroids were formed by forced floating method and were cultured for three days under static conditions prior to exposure to CYN. CYN influence on spheroid growth was measured daily and cell survival was determined by MTS assay and live/dead staining. The influence on cell proliferation, cell cycle alterations and induction of DNA damage (gH2AX) was determined using flow cytometry. Further, the expression of selected genes (qPCR) involved in the metabolism of xenobiotics, proliferation, DNA damage response, apoptosis and oxidative stress was studied. Results revealed that CYN dose-dependently reduced the size of spheroids and affected cell division by arresting HepG2 cells in G1 phase of the cell cycle. No induction of DNA double strand breaks compared to control was determined at applied conditions. The analysis of gene expression revealed that CYN significantly deregulated genes encoding phase I and II enzymes as well as genes involved in cell proliferation, apoptosis and DNA damage response. The advanced 3D HepG2 cell model due to its more complex structure and improved cellular interactions provides more physiologically relevant information and more predictive data for human exposure, and can thus contribute to more reliable genotoxicity assessment of chemicals including cyanotoxins.
COBISS.SI-ID: 19219971
The brain tissue landscape is very different from any other tissues and the specific microenvironment, comprising stem cells niches and blood-brain barrier, significantly influences the low rate of glioblastoma metastasis out of the brain, but better accommodates brain-invading cancer. This review addresses general cellular and molecular pathways that are to some extent similar in brain metastases and in glioblastoma, involving circulating tumor cells (CTCs) with cancer stem cells (CSCs) characteristics, and metastatic niches
COBISS.SI-ID: 5208399
The chemokine CCL5/RANTES is a versatile inflammatory mediator, which interacts with the receptor CCR5, promoting cancer cell interactions within the tumor microenvironment. Using immunohistochemistry, we identified CCL5 and CCR5 in a series of glioblastoma samples and cells, including glioblastoma stem cells. CCL5 and CCR5 mRNA levels were significantly higher in glioblastoma samples compare to non-cancerous brain tissues. The in vitro invasion of patients-derived primary glioblastoma cells and glioblastoma stem cells was dependent on CCL5-induced CCR5 signalling and is strongly inhibited by the small molecule CCR5 antagonist maraviroc. We have shown autocrine and paracrine crosstalk in glioblastoma and, in particular, glioblastoma stem cells with its stromal microenvironment, involve CCR5 and CCL5 and are contributing to glioblastoma invasion, suggesting the CCL5/CCR5 axis as a potential therapeutic target that can be targeted with repositioned drug maraviroc.
COBISS.SI-ID: 19416067
Due to increased frequency of cyanobacterial blooms and emerging evidence of cyanotoxicity in biofilm, reliable methods for early cyanotoxin threat detection are of major importance for protection of human, animal and environmental health. To complement the current methods of risk assessment, this study aimed to evaluate selected qPCR assays for detection of potentially toxic cyanobacteria in environmental samples. In the course of one year, 25 plankton and 23 biofilm samples were collected from 15 water bodies in Slovenia. Three different analyses were performed and compared to each other; qPCR targeting mcyE, cyrJ and sxtA genes involved in cyanotoxin production, LC-MS/MS quantifying microcystin, cylindrospermopsin and saxitoxin concentration, and microscopic analyses identifying potentially toxic cyanobacterial taxa. qPCR analyses detected potentially toxic Microcystis in 10 lake plankton samples, and potentially toxic Planktothrix cells in 12 lake plankton and one lake biofilm sample. A positive correlation was observed between numbers of mcyE gene copies and microcystin concentrations. Potential cylindrospermopsin- and saxitoxin-producers were detected in three and seven lake biofilm samples, respectively. The study demonstrated a potential for cyanotoxin production that was left undetected by traditional methods in both plankton and biofilm samples. Thus, the qPCR method could be useful in regular monitoring of water bodies to improve risk assessment and enable timely measures.
COBISS.SI-ID: 51505411