Anna Klimaszewska-Wiśniewska

Antiproliferative and antimetastatic action of quercetin on A549 non-small cell lung cancer cells through its effect on the cytoskeleton

To our knowledge, this study is the first to investigate the effect of the dietary flavonoid quercetin on the main cytoskeletal elements, namely microfilaments, microtubules and vimentin intermediate filaments, as well as cytoskeleton-driven processes in A549 non-small cell lung cancer cells. The methyl-thiazol-diphenyl-tetrazolium assay, annexin V/propidium iodide test, electron microscopic examination, cell cycle analysis based on DNA content, real-time PCR assays, in vitro scratch wound-healing assay, fluorescence staining of F-actin, β-tubulin and vimentin were performed to assess the effects of quercetin on A549 cells. Our results showed that quercetin triggered BCL2/BAX-mediated apoptosis, as well as necrosis and mitotic catastrophe, and inhibited the migratory potential of A549 cells. The disassembling effect of quercetin on microfilaments, microtubules and vimentin filaments along with its inhibitory impact on vimentin and N-cadherin expression might account for the decreased migration of A549 cells in response to quercetin treatment. We also suggest that the possible mechanism underlying quercetin-induced mitotic catastrophe involves the perturbation of mitotic microtubules leading to monopolar spindle formation, and, consequently, to the failure of cytokinesis. We further propose that cytokinesis failure could also be a result of the depletion of actin filaments by quercetin. These findings are important to our further understanding of the detailed mechanism of the antitumor activity of quercetin and render this flavonoid a potentially useful candidate for combination therapy with conventional antimicrotubule drugs, nucleic acid-directed agents or novel cytoskeletal-directed agents.

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.

Caffeine induces cytoskeletal changes and cell death in H1299 cells

Caffeine is the most common natural neuroactive substance around the world. The exact mechanism of the anticancer effects of caffeine is not clear, especially in the contexts of the cytoskeletal changes. It is known that caffeine exerts an effect on cell cycle, cell proliferation, radiosensivity of cells, and also induces cell death. The aim of the study was to determine the effect of 10 and 20 mM L−1 caffeine on the major cytoskeletal proteins in non-small lung cancer cell line H1299. Caffeine treatment induced abnormalities in morphology and ultrastructure of cells. Moreover, the fluorescence studies showed changes in organization of vimentin, β-tubulin, lamin A/C and F-actin, which were attributed to the induction of cell death. The results also demonstrated that caffeine induced formation of two cell populations: giant, mono- or multinucleated cells, with the phenotype of mitotic catastrophe and shrunken cells with condensation of chromatin, typical of apoptosis. This study for the first time shows the effect of caffeine on the cytoskeleton of H1299 cell line. In conclusion, a high-dose caffeine treatment induces apoptotic cell death and makes it a powerful anticancer agent that should be considered for the treatment of non-small cell lung cancer.

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.

Evaluation of Anti-Metastatic Potential of the Combination of Fisetin with Paclitaxel on A549 Non-Small Cell Lung Cancer Cells

The identification and development of new agents with a therapeutic potential as well as novel drug combinations are gaining the attention of scientists and clinicians as a plausible approach to improve therapeutic regimens for chemoresistant tumors. We have recently reported that the flavonoid fisetin (FIS), at physiologically attainable concentrations, acts synergistically with clinically achievable doses of paclitaxel (PTX) to produce growth inhibitory and pro-death effects on A549 human non-small cell lung cancer (NSCLC) cells. To further investigate a potential therapeutic efficacy of the combination of fisetin with paclitaxel, we decided to assess its impact on metastatic capability of A549 cells as well as its toxicity toward normal human lung fibroblast. Cell viability, cell migration, and invasion were measured by thiazolyl blue tetrazolium bromide (MTT) assay, wound healing assay, and Transwell chamber assay, respectively. The expression of metastasis-related genes was assessed with quantitative reverse transcriptase real-time polymerase chain reaction (qRT-PCR). Actin and vimentin filaments were examined under the fluorescence microscope. The combination of FIS and PTX significantly reduced cancer cell migration and invasion, at least partially, through a marked rearrangement of actin and vimentin cytoskeleton and the modulation of metastasis-related genes. Most of these effects of the combination treatment were significantly greater than those of individual agents. Paclitaxel alone was even more toxic to normal cells than the combination of this drug with the flavonoid, suggesting that FIS may provide some protection against PTX-mediated cytotoxicity. The combination of FIS and PTX is expected to have a synergistic anticancer efficacy and a significant potential for the treatment of NSCLC, however, further in vitro and in vivo studies are required to confirm this preliminary evidence.

THE INFLUENCE OF DOXORUBICIN ON NUCLEAR AND CYTOPLASMIC POOL OF F-ACTIN IN THE A549 CELL LINE

Cytoszkielet stanowi miedzykomorkowy system, odgrywający istotną role w prawidlowym funkcjonowaniu kazdej komorki. Jednym z jego komponentow jest F-aktyna, zaangazowana w zmiany ksztaltu, polarnośc, a takze ruch komorek. Celem przedstawionej pracy bylo określenie wplywu doksorubicyny na reorganizacje cytoszkieletu aktynowego oraz rodzaj indukowanej śmierci w komorkach linii A549. Wyniki badan oceniano przy uzyciu klasycznego oraz konfokalnego mikroskopu fluorescencyjnego, a takze z wykorzystaniem mikroskopii świetlnej oraz transmisyjnej mikroskopii elektronowej. W toku badan wykazano dawkozalezną wrazliwośc komorek linii A549 na doksorubicyne. Wraz ze wzrostem cytostatyku, wzrastal odsetek komorek martwych. Ponadto stwierdzono, ze doksorubicyna powoduje zmiany w reorganizacji F-aktyny w komorkach niedrobnokomorkowego raka pluca, a takze moze indukowac apoptoze oraz katastrofe mitotyczną. Dodatkowo potwierdzono wystepowanie aktyny na terenie jądra komorkowego.

Influence of arsenic trioxide on the expression of cyclin A in the Jurkat cel line

Improper expression of cell cycle regulators is characteristic for most cancer cells. Cyclins are a group of proteins, with concentration changing in time, which control progression through each phase of the cell cycle. Maximum cyclin A expression falls inside S and at the beginning of M phase. In the S phase cyclin A is responsible for a proper run of the replication process, and together with cyclin B conditions transition into the M phase. Arsenic trioxide (ATO) is an efficient drug used in acute promyelocytic leukemia (AML). Numerous reports point that arsenic trioxide can be applied in therapy of other types of cancer as well. The purpose of these study was to evaluate influence of arsenic trioxide on the expression and localization of cyclin A in Jurkat cell line. Additionally, we determined the effect of ATO on morphology and ultrastructure of the Jurkat cells. In order to reach this aim we applied classic light, florescence and electron transmission microscopy. A dose-dependent decrease in the percentage of viable cells was observed. Morphological and ultrastructural alterations suggest that apoptosis and autophagy are induced by ATO. Image-based cytometry showed high extent of necrotic cells after ATO treatment. A decrease in cyclin A level was observed, but without a change in protein nuclear localization.

Expression of cyclin A in A549 cell line after treatment with arsenic trioxide.

BACKGROUND Arsenic trioxide (ATO) is an effective drug used in acute promyelocytic leukemia (AML). Many reports suggest that ATO can also be applied as an anticancer agent for solid tumors in the future. The influence of arsenic trioxide on the expression of different cell cycle regulators is poorly recognized. The purpose of the current study is to investigate how arsenic trioxide affects cyclin A expression and localization in the A549 cell line. MATERIALS AND METHODS Morphological and ultrastructural changes in A549 cells were observed using light and transmission electron microscopes. Cyclin A localization was determined by immunofluorescence. Image-based cytometry was applied to evaluate the effect of arsenic trioxide on apoptosis and the cell cycle. Expression of cyclin A mRNA was quantified by real-time PCR. RESULTS After treatment with arsenic trioxide, increased numbers of cells with cytoplasmic localization of cyclin A were observed. The doses of 10 and 15 μM ATO slightly reduced expression of cyclin A mRNA. The apoptotic phenotype of cells was poorly represented, and the Tali imagebased cytometry analysis showed low percentages of apoptotic cells. The A549 population displayed an enriched fraction of cells in G0/G1 phase in the presence of 5μM ATO, whereas starting from the higher concentrations of the drug, i.e. 10 and 15 μM ATO, the G2/M fraction was on the increase. DISCUSSION Low expression of cyclin A in the A549 cell line may constitute a potential factor determining arsenic trioxide resistance. It could be hypothesized that the observed alterations in cyclin A expression/distribution may correlate well with changes in cell cycle regulation in our model, which in turn determines the outcome of the treatment.

Ultraviolet radiation (UV) induces f-action rearrangement and cell death in the A549 human lung cancer cell line

The human lung cancer cell line, A549, was employed to investigate the effect of UV radiation on the actin cytoskeleton organization in relation to its potential involvement in cell death processes. The light and electron microscopy analysis was performed to find the morphological signs of cell death in UV-treated A549 cells. In turn, the F-actin architecture was determined using fluorescence microscopy after phalloidin-Alexa Fluor 488 staining. We observed apoptosis as well as mitotic catastrophe-like morphological changes in A549 cells exposed to UV radiation. We also found the vacuoles in the cytoplasm of UV-exposed A549 cells, which are considered to be indicative of autophagy. All morphological effects of UV radiation were time-dependent. Furthermore, alterations in cell morphology corresponded with actin cytoskeleton reorganization. F-actin was presented in the form of dense ring-like structures surrounding the nuclei of cells with apoptotic-like phenotype. Moreover, in some of these cells depolymerization of F-actin occurred. On the other hand, the enlarged cells exhibited strongly expanded actin network. Our study revealed that ultraviolet radiation induced F-actin reorganization, which was accompanied by the characteristic apoptotic features. These results also suggest that UV induces not only the apoptosis but also the non-apoptotic cell deaths and in each of these processes reorganization of actin cytoskeleton is essential.

THE EFFECTS OF CAFFEINE ON CYTOSKELETON IN CHO AA8 CELL LINE

Kofeina to jeden z najcześciej spozywanych alkaloidow, spotykany miedzy innymi w napojach energetyzujących, kawie, herbacie czy czekoladzie, przez co stanowi ona skladnik codziennej diety milionow ludzi. Przez wiele lat uwaga naukowcow skupiala sie na dzialaniu kofeiny na organizmy zywe, w tym procesy fizjologiczne. Od niedawna, przedmiotem badan jest mechanizm dzialania kofeiny. Ze wzgledu na zaangazowanie bialek cytoszkieletu w procesy śmierci komorkowej, za zasadne uznano ocene indukowanych przez alkaloid zmian w obrebie wimentyny, F-aktyny oraz β-tubuliny. Ponadto analizowano morfologie i ultrastrukture komorek badanej linii. Podczas realizacji tematu wykorzystano metodyke z zakresu mikroskopii świetlnej, klasycznej mikroskopii fluorescencyjnej, a takze transmisyjnej mikroskopii elektronowej. Uzyskane wyniki wskazują, iz kofeina wplywa istotnie statystycznie na przezycie komorek linii CHO AA8. Obserwowane dwa typy morfologii komorek – obkurczonych, ze skondensowaną chromatyną, a takze olbrzymich z widocznymi mikrojądrami, sugeruje, iz alkaloid moze indukowac zarowno apoptoze, jak i katastrofe mitotyczną.