Rafał Krętowski

The Effects of Silica Nanoparticles on Apoptosis and Autophagy of Glioblastoma Cell Lines

Silica nanoparticles (SiNPs) are one of the most commonly used nanomaterials in various medical applications. However, possible mechanisms of the toxicity caused by SiNPs remain unclear. The study presented here provides novel information on molecular and cellular effects of SiNPs in glioblastoma LBC3 and LN-18 cells. It has been demonstrated that SiNPs of 7 nm, 5–15 nm and 10–20 nm induce time- and dose-dependent cytotoxicity in LBC3 and LN-18 cell lines. In contrast to glioblastoma cells, we observed only weak reduction in viability of normal skin fibroblasts treated with SiNPs. Furthermore, in LBC3 cells treated with 5–15 nm SiNPs we noticed induction of apoptosis and necrosis, while in LN-18 cells only necrosis. The 5–15 nm SiNPs were also found to cause oxidative stress, a loss in mitochondrial membrane potential, and changes in the ultrastructure of the mitochondria in LBC3 cells. Quantitative real-time PCR results showed that in LBC3 cells the mRNA levels of pro-apoptotic genes Bim, Bax, Puma, and Noxa were significantly upregulated. An increase in activity of caspase-9 in these cells was also observed. Moreover, the activation of SiNP-induced autophagy was demonstrated in LBC3 cells as shown by an increase in LC3-II/LC3-I ratio, the upregulation of Atg5 gene and an increase in AVOs-positive cells. In conclusion, this research provides novel information concerning molecular mechanisms of apoptosis and autophagy in LBC3 cells.

Hypoxia Enhances the Senescence Effect of Bortezomib—The Proteasome Inhibitor—On Human Skin Fibroblasts

The 26S proteasome inhibitor, bortezomib, selectively induces apoptosis in some cancer cells. However, the nature of its selectivity remains unknown. The study presented here provides novel information on cellular effects of bortezomib in normal fibroblasts. We have found that in normoxic conditions the percent of apoptotic cells did not change significantly, independently on incubation time and examined concentration of bortezomib (25 nmol/L or 50 nmol/L). In hypoxic conditions we did not observe any effect of bortezomib on apoptosis of fibroblasts incubated for 24 h and 48 h in comparison to control. Only in the case of fibroblasts incubated for 12 hours in hypoxia significant increase in apoptosis, dependent on concentration of bortezomib, was observed. Our study has shown that bortezomib causes a time-dependent increase in senescence of normal fibroblasts, especially of these incubated in hypoxic conditions. Moreover, we demonstrated that oxygen regulated protein 150 (ORP150) expression was induced in fibroblasts in hypoxia conditions only, suggesting that this protein may play an important role in the cytoprotective response to environmental stress.

The profile of lysosomal exoglycosidases in replicative and stress-induced senescence in early passage human fibroblasts

The aim of the present study was to assess the profiles of the exoglycosidases: N-acetyl-β-hexosoaminidase, β glucuronidase and β galactosidase, α mannosidase and α fucosidase in fibroblast culture undergoing replicative and stress-induced senescence. Half of the cell culture was grown in normal conditions, without the stressor, and the other half of the cell was treated with 0.15 mM tert-butylhydroperoxide. The activities of total N-acetyl-β-hexosoaminidase as well as β glucuronidase in the cell lysate were determined in duplicates using the method of Marciniak et al. The activities of β galactosidase, α mannosidase and α fucosidase in the cell lysate were determined in duplicates using the method of Chatteriee et al. with the modification by Zwierz et al. The activities of the exoglycosidases examined, with the exception of β glucuronidase, showed a significant increase between individual days of the experiment in both non-stressed and stressed fibroblast cell culture. On each day of the experiment, in the cell lysate of stressed fibroblasts, the activities of exoglycosidases were significantly higher compared to the non-stressed cells. There were very strong correlations between SA-β-GAL staining and b galactosidase activity on individual days of the experiment in both non-stressed and stressed fibroblast cell culture. Replicative and stress-induced senescence results in significant changes to the level of lysosomal exoglycosidases, and results in enhanced lysosomal degradative capacity.

Metronidazole Decreases Viability of DLD-1 Colorectal Cancer Cell Line

The aim of our study was to evaluate the impact of metronidazole (MTZ) on DLD-1 colorectal cancer cell (CRC) line. Toxicity of MTZ was determined by MTT test. Cells were incubated with MTZ used in different concentrations for 24, 48, and 72 hours. The effect of MTZ on DNA synthesis was measured as [3H]-thymidine incorporation. The morphological changes in human DLD-1 cell line were defined by transmission electron microscope OPTON 900. The influence of MTZ on the apoptosis of DLD-1 cell lines was detected by flow cytometry and fluorescence microscopy, while cell concentration, volume, and diameter were displayed by Scepter Cell Counter from Millipore. Our results show that cell viability was diminished in all experimental groups in comparison with the control, and the differences were statistically significant. We did not find any significant differences in [3H]-thymidine incorporation in all experimental groups and times of observation. Cytofluorimetric assays demonstrated a statistically significant increase of apoptotic rate in MTZ concentrations 10 and 50 μg/mL after 24 hours; 0.1, 10, 50, and 250 μg/mL after 48 hours; and in all concentrations after 72 hours compared with control groups. In the ultrastructural studies, necrotic or apoptotic cells were occasionally seen. In conclusion, MTZ affects human CRC cell line viability. The reduction of cell viability was consistent with the apoptotic test.

Possible Mechanisms of the Prevention of Doxorubicin Toxicity by Cichoric Acid—Antioxidant Nutrient

Skin is the largest organ in the human body, and which protects organism against unfavorable external factors e.g., chemicals, environment pollutants, allergens, microorganisms, and it plays a crucial role in maintaining general homeostasis. It is also an important target of oxidative stress due to the activity of oxygen reactive species (ROS), which are constantly generated in the fibroblasts in response to exogenous or endogenous prooxidant agents. An example of such compound with proved prooxidant activity is Doxorubicin (DOX), which is an effective anticancer agent belongs in anthracycline antibiotic group. Increasingly frequent implementation of various strategies to reduce undesirable DOX side effects was observed. Very promising results come from the combination of DOX with dietary antioxidants from the polyphenol group of compounds, such as cichoric acid (CA) in order to lower oxidative stress level. The aim of this work was to evaluate the influence of CA combined with DOX on the oxidative stress parameters in fibroblasts, which constitute the main cells in human skin. We also wanted to examine anti-apoptotic activity of CA in fibroblasts treated with selected concentrations of DOX. Results obtained from the combination of DOX with CA revealed that CA exhibits cytoprotective activity against DOX-induced damage by lowering oxidative stress level and by inhibiting apoptosis. The present finding may indicate that CA may serve as antioxidative and anti-apoptotic agent, active against DOX-induced damage.

Glucose deficiency reduces collagen synthesis in breast cancer MCF7 cells

We decided to study the effect of glucose deprivation on collagen metabolism in MCF7 cells. The incorporation of [3H]‐proline into collagenase‐sensitive and hydroxyproline‐containing proteins was used as an index of collagen synthesis, whereas pulse—chase technique was employed to evaluate the degradation of newly synthesized proteins. The MCF7 cells incubated in high glucose medium synthesized detectable amounts of collagenous proteins. Most of them were found in the cell layer. The shortage of glucose resulted in about 30% reduction in collagen synthesis. The pulse—chase experiments demonstrated that proportionally less collagen was degraded in cultures incubated in low‐glucose than in high‐glucose media.

Silica nanoparticle-induced oxidative stress and mitochondrial damage is followed by activation of intrinsic apoptosis pathway in glioblastoma cells

Introduction Recently, the focus of oncological research has been on the optimization of therapeutic strategies targeted at malignant diseases. Nanomedicine utilizing silicon dioxide nanoparticles (SiNPs) is one such strategy and is rapidly developing as a promising tool for cancer diagnosis, imaging, and treatment. Nevertheless, little is known about the mechanisms of action of SiNPs in brain tumors. Materials and methods Here, we explored the effects of 5–15 nm SiNPs in the human glioblastoma cell line LN229. In this respect, MTT assays, microscopic observations, flow cytometry analyses, and luminescent assays were performed. Moreover, RT-qPCR and Western blot analyses were done to determine gene and protein expressions. Results We demonstrated that SiNPs triggered evident cytotoxicity, with microscopic observations of the nuclei, annexin V–fluorescein isothiocyanate/propidium iodide staining, and elevated caspase 3/7 activity, suggesting that SiNPs predominantly induced apoptotic death in LN229 cells. We further showed the occurrence of oxidative stress induced by enhanced reactive oxygen-species generation. This effect was followed by deregulated expression of genes encoding the antioxidant enzymes SOD1, SOD2, and CAT, and impaired mitochondria function. SiNP- induced mitochondrial dysfunction was characterized by membrane-potential collapse, ATP depletion, elevated expression of BAX, PUMA, and NOXA with simultaneous downregulation of BCL2/BCL2L1, and activation of caspase 9. Moreover, RT-qPCR and Western blot analyses demonstrated increased levels of the endoplasmic reticulum stress markers GRP78, GRP94, and DDIT3, as well as strongly increased expressions of the IL1B and COX2 genes, suggesting activation of endoplasmic reticulum stress and a proinflammatory response. Conclusions Altogether, our data indicate that in LN229 cells, SiNPs evoke cell death via activation of the intrinsic apoptosis pathway and suggest that other aspects of cellular function may also be affected. As such, SiNPs represent a potentially promising agent for facilitating further progress in brain cancer therapy. However, further exploration of SiNP long-term toxicity and molecular effects is necessary prior to their widespread application.

The effect of quercetin on the electrical properties of model lipid membranes and human glioblastoma cells

Quercetin is a naturally-occurring flavonoid claimed to exert many beneficial health effects. In this report, the influence of quercetin on the surface charge of phosphatidylcholine liposomes and human glioblastoma LN-229 and LN-18 cells was studied using microelectrophoretic mobility measurements. The effect of quercetin on the electrical resistance and capacitance of bilayer lipid membranes was analyzed via electrochemical impedance spectroscopy. The results showed that after flavonoid treatment, the cell lines demonstrated changes in surface charge only in alkaline pH solutions, whereas there were no significant alterations in quercetin-treated vs. control cells in acidic pH solutions. The same tendency was found for liposomal membranes proving that quercetin insertion into membranes is strongly pH-dependent. Capacitance and resistance measurements conducted in acidic electrolyte solutions demonstrated an increase in both electrical parameters, indicating an increased amount of quercetin inserted into the bilayers. Moreover, the cytotoxic effect of quercetin confirms that the flavonoid enters the cells and perturbs the proliferation of LN-229 and LN-18 glioblastoma cell lines. As such, our results indicate that the specific localization of quercetin, membrane-bound or cell-entering, might be crucial for its pharmacological activity. However, further studies are necessary prior to applying these physicochemical measurements as standard methods of evaluating drug activity.