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Background: The extent of morphological and ultrastructural changes in HeLa cells was assessed by optical, fluorescence and electron microscopy after exposure to various concentrations of physcion, taking into account the biological properties of the test compound. Methods: Cell viability was assessed by MTT assay, while the cell cycle, LC3 expression, apoptosis, change of mitochondrial potential, Bcl-2 protein expression level and the level of reactive oxygen species were analyzed by flow cytometry. Results: As a result of physcion encumbrance, concentration-dependent inhibition of HeLa cell viability and the G0/G1 phase of the cell cycle was observed. Activation of the lysosomal system was also revealed, which was expressed by an increased number of lysosomes, autophage vacuoles and increased expression of the LC3 protein, a marker of the autophagy process. Transmission electron microscopy and fluorescence microscopy showed that physcion induced clear changes in cervical cancer cells, especially in the structure of the nucleus and mitochondria, which correlated with the production of reactive oxygen species by the test compound and indicated the induction of the oxidative process. At the same time, the pro-apoptotic effect of physcion was demonstrated, and this mechanism was dependent on the activation of caspases 3/7 and the reduction in Bcl-2 protein expression. Conclusion: The obtained results indicate an antitumor mechanism of action of physcion, based on the induction of oxidative stress, autophagy and apoptosis.

Background Anthraquinones, including emodin, are compounds with numerous pharmacological properties, including anticancer properties. The aim of this study experiment was to examine the effect of emodin, a natural compound present in the roots and rhizomes of Rheum palmatum, on the induction of mitotic catastrophe in cervical cancer cells. Material and methods HeLa celIs were treated with different emodin concentrations for 48 h, and cell growth was measured with 3-(4-,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolyl. The cell-cycle distribution and the level of apoptosis were determined by means of flow cytometry, using annexin V-fluorescein isothiocyanate staining and propidium iodide. Morphological changes in the mitotic apparatus were evaluated using optical and confocal microscopy techniques. Results Emodin induced an increase in the number of polymorphonuclear cells, giant cells, cells with micronuclei, cells with abnormal mitosis and damaged spindle. The reorganization of F-actin depended on the concentration of emodin. With the increase in emodin concentration, inhibition of mitotic activity was demonstrated, which was manifested by a decrease in the mitotic index, mainly in metaphase of the mitotic process and an increase in the number of cells inhibited in the G2/M phase. At the same time, an increase in the number of apoptotic cells was found. Conclusion Emodin leads to death of cervical cancer cells by induction of a mitotic catastrophe.

Melatonin is a hormone with many different biological activities and therefore seems to be an important factor reducing the harmful effects caused by toxic organophosphorus compounds. In this study, we attempted to evaluate the protective effect of melatonin on liver cells of mice challenged with chemical warfare agent-soman. The study was conducted at the level of ultrastructural and biochemical changes (analysis of the activity of model lysosomal enzymes and assessment of the level of lipid peroxidation). Significant biochemical and ultrastructural changes were found in the studied mouse hepatocytes after administration of soman alone, and soman in combination with melatonin, and the scope of the disclosed changes was dependent on the time of action of the examined factors. Melatonin has shown protective action, shielding liver cells from toxic effects of soman, which may result from its antioxidant properties and stimulation of the lysosomal compartment, the system coordinating the isolation and removal of cell-threatening processes.

Chrysophanol is an anthraquinone with proven antitumor activity against several tumor cell lines. However, its effect on cervical cancer cells is still unknown. Therefore, HeLa cells were exposed to various concentrations of chrysophanol and then subjected to biochemical, ultrastructural, and morphological analysis. It has been shown using flow cytometry and MTT reduction assay that chrysophanol has been shown to inhibit cell viability and arrest cells in the G2/M phase of the cell cycle. Using Annexin V/propidium iodide staining, a significant increase in apoptosis was found after chrysophanol treatment on HeLa cells, and this process was mediated by caspases 3/7 with a clear inactivation of the antiapoptotic Bcl-2 family protein. However, the demonstrated increased number of cells with double-stranded DNA breaks suggests that chrysophanol also causes DNA damage. By means of electron and fluorescence microscopy, a clear effect of chrysophanol on the intensification of degradation processes, on changes in the structure of the nucleus, endoplasmic reticulum and mitochondria was demonstrated. The changes visible in the mitochondria may be related to the increase in the level of free radicals induced by chrysophanol, which induces apoptosis, inter alia, by increasing the permeability of mitochondrial membranes. The range of observed changes depended on the concentration of anthraquinone was tested.

Although vitamin D is included in the group of fat-soluble vitamins, it must be considered as a prohormone. Its active forms including calcitriol have pleiotropic effects and play an important role in the regulation of cell proliferation, differentiation and apoptosis as well as in hormone secretion, and they show anti-cancer properties. Since calcitriol delivery can be beneficial for the organism, and Syrian golden hamsters represent a unique experimental model, we decided to investigate its toxicity in this species. In this study, we injected calcitriol intraperitoneally at doses 0 (control), 0.180±0.009 µg/kg and 0.717± 0.032 µg/kg. Animal behavior was observed for 72 hrs after injection, and afterwards blood and liver and kidney were collected for post-mortem examination, electron microscopy, and hematology analyses. The highest dose of calcitriol induced a change in animal behavior from calm to aggressive, and liver surface showed morphological signs of damage. Following the injection of calcitriol, ultrastructural changes were also observed in the liver and kidneys, e.g. vacuolization and increased number of mitochondria. There was also a trend for increased serum levels of aspartate aminotransferase (AST), but not of alanine aminotransferase (ALT) or GGTP (gamma-glutamyl transpeptidase). There was no change in Ca, Mg and P levels as well as in blood morphology between experimental and control groups. These results indicate that calcitriol at 0.717, but not at 0.18 µg/kg, may induce acute damage of the liver and kidneys, without inducing calcemia. We propose that the hepatotoxic effect of calcitriol in hamster constitutes the primary cause of behavioral changes.

The aim of this study was to evaluate ultrastructural changes in kidney and liver tissue of chicken embryos exposed in ovo to cadmium. Embryonated eggs were injected on the 4th day of incubation with cadmium at the dose of 0, 2, 4 and 8 μg/egg (80 eggs/group). The samples of kidney and liver tissues were collected from embryos at the 14th and 18th day of incubation (E14 and E18) and at hatching day (D1). The tissue structure was evaluated by transmission electron microscopy (Tecnai G2 Spirit). The results indicate that hepatocytes responded to damage caused by toxic cadmium activity with a significant disturbance in the structure of mitochondria and a considerable expansion of the lysososmal system, while glomerular cells additionally reacted with an increased proliferation of peroxisomes. The range of changes observed on the subcellular level was dependent on the dose of cadmium, embryogenesis stage and cell type.

Background Aloe-emodin is an anthraquinone with potential pharmacological properties, including numerous antitumor properties. The purpose of the study was to determine whether aloe-emodin induces mitotic death in cervical cancer cells. Materials and methods Analysis of morphological changes as surrogate mitotic death indicators in HeLa cells was carried out using optical, fluorescence and electron microscopy. Viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide reduction assay. Cell-cycle analysis was performed using flow cytometry. Results Aloe-emodin increased the number of multinucleate cells, giant and micronuclear cells. There was a concentration-dependent decrease in the mitotic index with a predominance of cells in the metaphase of the mitotic process and inhibition of division in the G2/M phase of the cell cycle. The presence of cells with abnormal mitosis and cells with injury to the division spindle was also demonstrated. Conclusion Aloe-emodin induces mitotic catastrophe in cervical cancer cells.

Background: Aloe-emodin belongs to the group of anthraquinones having extremely high biological activity. The aim of this study was to evaluate the range of morphological and biochemical changes in HeLa cells treated with aloe-emodin, especially with regard to the lysosomal compartment. Methods: Marking of lysosomes was performed with neutral red staining for conventional light microscopy and acridine orange staining for confocal microscopy. To evaluate ctivity of lysosomal enzymes and permeability of the lysosomal membrane, spectrophotometric techniques were employed. Results: Aloe-emodin caused increased permeability of lysosomal membranes in HeLa cells, expressed inter alia by extinction of the orange color of acridine orange (lysosomal marker) and in reduction of neutral red uptake by lysosomes. These changes are accompanied by release of cathepsins from the interior of the lysosomes with a simultaneous highly significant increase in their activity in the cytoplasm. Conclusion: The results indicate that aloe-emodin can activate lysosomal pathway-dependent apoptosis in HeLa cells.

BACKGROUND/AIM To search for new antileukemic agents, the chemical structure of phenytoin was modified. A possible cytotoxic activity of three bromoalkyl phenytoin analogs, methyl 2-(1-(3-bromopropyl)-2,4-dioxo-5,5-diphenylimidazolidin-3-yl) propanoate (PH2), 1-(3-bromopropyl)-3-methyl-5,5-diphenylimidazolidine-2,4-dione (PH3) and 1-(4-bromobutyl)-3-methyl-5,5-diphenylimidazolidine-2,4-dione (PH4) on regulated cell death, the cell cycle and cell ultrastructure was assessed. MATERIALS AND METHODS The experiments were performed in vitro on HL-60 and U937 cells, using flow cytometry and electron microscopy methods. RESULTS Application of PH2, PH3, and PH4 resulted in cell surface exposure of phosphatidylserine and plasma membrane impairment, caspase-8, -9, and -3/7 activation, dissipation of mitochondrial membrane potential, DNA breakage, cell-cycle disturbance and cell ultrastructural changes. In general, PH3 appeared to be the most active against the leukemia cells, and all bromoalkyl hydantoins, PH2-PH4, were more active in HL-60 cells than in U937 cells. CONCLUSION The antileukemic activity of the bromoalkyl phenytoin analogs depended on the combination of N-hydantoin substituents and the human cell line used.

Background An example of plant-derived compounds that may be of great importance in oncological therapy is emodin. The aim of this study was to evaluate the range of changes in the lysosomal compartment of HeLa cervical cancer cells treated with emodin. Materials and methods Changes in the lysosomal compartment were evaluated using microscopic techniques. In order to evaluate the activity of lysosomal enzymes and the permeability of the lysosomal membrane, a spectrophotometric technique was employed. Results Emodin induced significant changes in the lysosomal compartment, manifesting as an increase in the number of lysosomes, autophagic vacuoles and the activity of lysosomal hydrolases. Emodin exhibited cytotoxic activity against lysosomes through lysosomal membrane damage with possible leaks of lysosomal hydrolases into the cytoplasm. Conclusion Emodin induces degradation processes and promotes the death of tumor cells through a mechanism that occurs with clear involvement of the lysosomal compartment.