Gelsolin is a potential cellular target for cotinine to regulate the migration and apoptosis of A549 and T24 cancer cells.

In the present work, we have investigated the effect of cotinine, the major metabolite of nicotine on the A549 and T24 cell lines in the context of structural and quantitative changes of F-actin, gelsolin and vimentin. The chosen cell lines constitute the established experimental models for lung and bladder cancers, respectively, in the case of which, smoking cigarettes is one of the key factor increasing their incidence rate significantly. In order to evaluate the impact of cotinine on the viability and proliferation of A549 and T24 cells, the MTT assay was performed. The organization and distribution of F-actin, gelsolin and vimentin were examined using conventional and confocal fluorescence microscopy. The levels of F-actin and gelsolin as well as the percentages of apoptotic and dead cells were assessed using the image-based cytometer. The ultrastructural changes of cotinine-treated A549 and T24 cells were visualized under the transmission electron microscopy. We have shown here that cotinine enhances the survival and proliferation rate of A549 and T24 cells. We have also found that in A549 cells, but not in T24 cell line, cotinine acted stimulating on the vimentin filament network. Furthermore, the increase in the fluorescence intensity of gelsolin upon the addition of cotinine to the T24 cells was found to be correlated with the lack of apoptosis induction as well as the increase of migration potential of these cells. On the other hand, the cotinine-induced decrease in the fluorescence intensity of gelsolin was associated with the increase in the percentages of apoptotic A549 cells and the decreased migratory ability of these cells. Based on the obtained results, we propose that the gelsolin is an important cellular target for cotinine, through which this compound influences on the basic processes involved in neoplastic transformation and metastasis, such as migration and apoptosis.

Expression of cyclin D1 after treatment with doxorubicin in the HL-60 cell line.

Increased levels of cyclin D1 and amplification of CCND1 gene occur in many types of cancers. We have followed the expression of cyclin D1 after treatment with doxorubicin with reference to cell death and other possible therapeutic implications. The effect of the treatment on the cell cycle, survival, intracellular level (flow cytometry), and intracellular localization of cyclin D1 (fluorescence microscopy) and expression of CCND1 (real-time RT-PCR) was investigated in HL-60 cells. An increase in the fluorescence intensity of cyclin D1 occurred after treatment with 0.15 and 0.3 µM doxorubicin. This tendency was confirmed by real-time RT-PCR. Expression of CCND1 in relation to the reference gene PBGD was increased in cells exposed to 0.15 µM doxorubicin. Concomitantly, some alterations in the regulation of the G0/G1, S, and G2/M checkpoints occurred, accompanied by changes in the polyploid fraction of the population. This was particularly evident at 0.3 µM doxorubicin, at which concentration the rate of cell death was also clearly higher. In conclusion, depending on the concentration used, alterations in cell death and the number of S, G2/M, and polyploid cells may correspond with cyclin D1 levels. This, in turn, may reflect an important role of the protein as one of the possible survival/point-of-no-return regulators dependent on its concentration, which seems especially plausible in the context of more prominent cell death in the above-mentioned fractions of cells.

[Involvement of Rho proteins in G1 phase cell cycle progression]. / Udzial bialek Rho w regulacji postepu fazy G1 cyklu komórkowego.

 Rho proteins, including RhoA, Rac1 and Cdc42, are members of the Ras superfamily of monomeric GTP-binding proteins, which are well-known regulators of the cytoskeleton. Numerous studies have shown that an intact cytoskeleton is required for cell cycle progression through G1 phase as well as mitosis and cytokinesis. Because of their role in both cytoskeletal rearrangement and mitogenic signaling, Rho family proteins are key mediators of cell cycle progression. In this paper, we review the current state of knowledge concerning the Rho-dependent signaling pathways that regulate the expression of cell cycle regulatory proteins required for G1 phase progression and S phase entry.

Hyperthermia induces cytoskeletal alterations and mitotic catastrophe in p53-deficient H1299 lung cancer cells.

Hyperthermia is used in cancer therapy, however much remains to be discovered regarding its mechanisms of action at the cellular level. In this study, the effects of hyperthermia on cell death, survival, morphology and the cytoskeleton were investigated in a non-small cell lung cancer cell line, H1299. Despite the fact that this cell line is widely used in research, it has not yet been tested for heat shock sensitivity. Cells were given a 30-min heat shock at 43.5°C and 45°C and left to recover at 37°C for 24 and 48 h. 24 h after heat shock treatment, we monitored changes in the organization of the cytoskeleton using immunofluorescence microscopy. The number of actin stress fibers was significantly reduced, microtubules formed a looser meshwork, a portion of the cells possessed multipolar mitotic spindles, whereas vimentin filaments collapsed into perinuclear complexes. 48 h following heat stress, most of the cells showed recovery of the cytoskeleton, however we observed a considerable number of giant cells that were multinucleated or contained one enlarged nucleus. The data obtained by MTT assay showed a dose-dependent decrease of cell viability, while flow cytometric analysis revealed an increase in the number of cells with externalized phosphatidylserine. The results suggest that one of the modes of heat-induced cell death in H1299 cells is mitotic catastrophe, which probably ends in apoptosis.

Ciprofloxacin is a potential topoisomerase II inhibitor for the treatment of NSCLC.

Lung cancer is one of the most common tumors and its treatment is still inefficient. In our previous work we proved that ciprofloxacin has a different influence on five cancer cell lines. Here, we aimed to compare the biological effect of ciprofloxacin on cell lines representing different responses after treatment, thus A549 was chosen as a sensitive model, C6 and B16 as highly resistant. Three different cell lines were analyzed (A549, B16 and C6). The characterization of continuous cell growth was analyzed with the Real-Time Cell Analyzer (RTCA)-DP system. Cytoskeletal changes were demonstrated using immunofluorescence. The cell cycle was analyzed using flow cytometry. Ciprofloxacin was cytostatic only against the A549 cell line. In the case of other tested cell lines a cytostatic effect was not observed. Cytoskeletal analysis confirms the results obtained with RTCA-DP. A549 cells were inhibited in the G2/M phase suggesting a mechanism related to topoisomerase II inhibition. The biological effects of ciprofloxacin support the hypothesis that this drug can serve as an adjuvant treatment for lung cancer, due to its properties enabling topoisomerase II inhibition.

[Cotinine--metabolism, application as a biomarker and the effects on the organism]. / Kotynina--metabolizm, zastosowanie jako biomarker i wplyw na organizm czlowieka.

This review presents the current state of knowledge on cotinine, the major metabolite of nicotine. Special attention is paid to the formation of this compound in the organism, its metabolism, application in diagnostic procedures and evaluation of its in vitro and in vivo activities. For many years, cotinine has been used as a biomarker of exposure to tobacco smoke. Currently, this compound is applied in many other studies including the use of cotinine in the treatment of various diseases. Several years ago, Scott et al. patented therapeutic applications of cotinine in chronic and acute inflammation. Cotinine is an interesting compound with a well-known metabolism; therefore there are suggestions for its application in the diagnosis and treatment of certain diseases. 

[Rho proteins - the key regulators of cytoskeleton in the progression of mitosis and cytokinesis]. / Bialka Rho − kluczowe regulatory cytoszkieletu w przebiegu mitozy i cytokinezy.

The Rho proteins are members of the Ras superfamily of small GTPases. They are thought to be crucial regulators of multiple signal transduction pathways that influence a wide range of cellular functions, including migration, membrane trafficking, adhesion, polarity and cell shape changes. Thanks to their ability to control the assembly and organization of the actin and microtubule cytoskeletons, Rho GTPases are known to regulate mitosis and cytokinesis progression. These proteins are required for formation and rigidity of the cortex during mitotic cell rounding, mitotic spindle formation and attachment of the spindle microtubules to the kinetochore. In addition, during cytokinesis, they are involved in promoting division plane determination, contractile ring and cleavage furrow formation and abscission. They are also known as regulators of cell cycle progression at the G1/S and G2/M transition. Thus, the signal transduction pathways in which Rho proteins participate, appear to connect dynamics of actin and microtubule cytoskeletons to cell cycle progression. We review the current state of knowledge concerning the molecular mechanisms by which Rho GTPase signaling regulates remodeling of actin and microtubule cytoskeletons in order to control cell division progression.

The influence of cotinine on the non-small-cell lung cancer line A549.

The aim of this investigation was to determine the influence of cotinine on the non-small-cell lung cancer line A549. The material for the study was the A549 cell line. The cells were subjected to 24-h incubation with cotinine at doses of 18 and 36 ng/ml. Control cells were incubated under analogous conditions without the addition of cotinine. Cell viability was determined and the cells were Mayer hematoxylin stained, embedded in epon, and observed under an electron microscope. A fluorescent method was used to evaluate F-actin. Cotinine's action resulted in alteration of the cytoskeleton, with changes in the organization of F-actin. Also observed was A549 cell death, presumably due to the activation of apoptotic and mitotic catastrophe pathways. Moreover, the results at the ultrastructural level suggest that cotinine influences not only the nucleus and cell shape by acting on the actin cytoskeleton, but also plays a crucial role in changes which include the remaining cellular organelles. Until now, research has been focused on determining the action of constituents of cigarette smoke, mainly nicotine. From this point of view, studies on cotinine seem justified. Results of the study allow us to suppose that cotinine, through F-actin, can influence the size and shape of non-small-cell lung cancer cells. There are some suggestions that cells of the A549 cell line can undergo death through apoptotic and mitotic catastrophe pathways as a result of cotinine's action.

[The Rho protein family and its role in the cellular cytoskeleton]. / Rodzina bialek Rho i ich rola w cytoszkielecie komórki.

Proteins of the Rho protein family (RhoA, Rac1, Cdc42) work as molecular switches in the regulation of many cellular processes. They are involved in cell migration, cell-cycle control, apoptosis, and the regulation of gene transcription. Rho proteins show their activity mainly in the cell's cytoskeleton by taking part in the reorganization of microfilaments and microtubules. In recent years, significant progress has been made in understanding the biochemical and genetic nature of many processes controlled by Rho proteins. Although there are still seveal unknowns and hypothesizes which require confirmation, newer and more precise experimental results allow us to better understand these processes. It has also been suggested to use Rho family proteins in the therapy of some diseases. The purpose of this study was to characterize the significance of Rho processes in the cellular cytoskeleton.