The alterations in SATB1 and nuclear F-actin expression affect apoptotic response of the MCF-7 cells to geldanamycin

Introduction. The function and localization of actin in the nucleus have not yet been fully described. However, actin seems to be a key protein in nuclear processes interacting with chromatin and matrix proteins. The aim of the study was to evaluate the effect of controlled expression of nuclear pool of F-actin and special AT-rich sequence-binding protein 1 (SATB1) on the in vitro induction of active cell death by geldanamycin (GA). Material and methods. The expression of SATB1 was regulated by the transfection of non-aggressive breast cancer MCF-7 cells with siRNA against SATB1 or expression plasmid with cloned cDNA of SATB1. The altered expression of cofilin-1 in these cells was used to regulate the nuclear expression and localization of F-actin. The effect of GA was analyzed in the context of cell death induction and cell cycle alterations. Results. Our studies revealed that the targeted regulation of SATB1 and cofilin-1 expression changed the apoptotic response of the MCF-7 cells to GA. The overexpression of these proteins potentiated GA-induced arrest of the cells in the G1 phase of cell cycle and increased the population of the hypodiploid cells. Conclusion. The alterations in the nuclear expression of SATB1 and F-actin in MCF-7 cells may affect their active cell death in response to GA

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.W niniejszej pracy użyto linii komórkowej ludzkiego raka płuca, A549, w celu określenia wpływu promieniowania UV na organizację cytoszkieletu aktynowego w powiązaniu z jego potencjalnym udziałem w śmierci komórki. Wykonano analizy na poziomie mikroskopu świetlnego i elektronowego, które umożliwiły określenie zmian morfologicznych wskazujących na indukcję różnych rodzajów śmierci komórkowej pod wpływem promieniowania UV. Filamenty aktynowe wyznakowane zostały falloidyną skoniugowaną z fluorochromem Alexa Fluor 488. Po ekspozycji komórek na działanie promieniowania UV obserwowano liczne komórki morfologicznie podobne do apoptotycznych, jak również te, o cechach morfologicznych charakterystycznych dla katastrofy mitotycznej. W komórkach linii A549 traktowanych promieniowaniem UV zaobserwowano również liczne wakuole cytoplazmatyczne, które są uważane za wyznacznik autofagicznej śmierci komórek. Wszystkie zaobserwowane zmiany były zależne od czasu ekspozycji i korelowały z reorganizacją cytoszkieletu aktynowego. W komórkach apoptotycznych F-aktyna występowała w postaci pierścieni otaczających jądra komórkowe. Ponadto, w niektórych komórkach o fenotypie apoptozy F-aktyna uległa depolimeryzacji. Z drugiej strony komórki powiększone wykazywały znaczne rozbudowanie sieci filamentów aktynowych. Prezentowane badania wskazują na reorganizację cytoszkieletu aktynowego po ekspozycji komórek na promieniowanie UV, która wiąże się głównie z procesem apoptozy. Jednakże, wyniki doświadczeń sugerują, że promieniowanie UV indukuje także nieapoptotyczne rodzaje śmierci komórek, w przebiegu których reorganizacja F-aktyny jest również istotna

The role of exportin 6 in cytoskeletal-mediated cell death and cell adhesion in human non-small-cell lung carcinoma cells following doxorubicin treatment

The actin cytoskeleton plays an important role in various cellular processes. The different forms ofactin (G-actin and F-actin) participate in the organization of nuclear structure and its functions. The structure of the actin cytoskeleton is controlled by proteins involved in the translocation of actin between cytoplasm and the nucleus. In this study, we used siRNA method to investigate the role of exportin 6 in the switching between nuclear and cytoplasmic F-actin pools in H1299 cells treated with no, 1.0 or 2.5 μM doxorubicin. We showed that silencing of exportin 6 expression changed the response of H1299 to doxorubicin. Here, we observed increased population of cells affected by doxorubicin-induced necrotic cell death. Furthermore, fluorescence studies showed that downregulation of exportin 6 exerted profound DOX-induced changes in the F-actin cytoskeleton architecture. The F-actin cytoskeleton was seen in the form of small fibers or aggregates after doxorubicin treatment. Additionally, some cells lost cell adhesion properties. Downregulation of exportin 6 influenced also transcriptional activity of the cells. In cells transfected with nontargeting siRNA, we observed a higher level of 5’-fluorouridine fluorescence than in cells with silenced export in 6 expression. In conclusion, we showed that downregulation of exportin 6 induced necrotic cell death. Moreover, the observed alterations of cell adhesion suggest the key role of cytoplasmic F-actin in maintaining intercellular junctional complexes and/or focal adhesion properties and the importance of the balance between nuclear and cytoplasmic F-actin pools

Nornicotine impairs endothelial cell-cell adherens junction complexes in EA.hy926 cell line via structural reorganization of F-actin

The aim of the study was to estimate the effect of nornicotine on endothelial EA.hy926 cells in the context of its impact on cell-cell junctions. The objective of the study was to determine the relationship between junctional proteins and F-actin after treating the cells with nornicotine. After 24 h of cell exposure to 0.08, 0.12, and 0.16 ng/mL nornicotine, analysis was performed of cell death, cell migration, ultrastructure, and colocalization of beta-catenin/F-actin and zonula occludens (ZO)-1/F-actin. Our study did not reveal any alterations in EA.hy926 cell line survival following treatment with nornicotine. However, nornicotine exerted disparate effects on cell migration and led to changes in both the ultrastructure and organization of cell-cell junctional complexes and F-actin. Moreover, the cell migration observed in the experiments performed in the present work negatively correlated with the number of Weibel-Palade bodies seen through transmission electron microscopy (TEM). Moreover, the mechanism of cell migration promotion was VEGF-independent, and the decrease in the number of Weibel-Palade bodies resulted from nornicotine-induced F-actin depolymerization. In conclusion, the present study demonstrated that low concentrations of nornicotine do not affect cell survival, but promote cell movement and impair adherens junctions through changes in F-actin organization. Our results indicate for the first time the effect of nornicotine on endothelial EA.hy926 cells and suggest that nornicotine may induce transmigration pathways and, consequently, facilitate the transendothelial migration of monocytes associated with atherosclerosis

Effect of arsenic trioxide (Trisenox) on actin organization in K-562 erythroleukemia cells.

Actin is one of the cytoskeletal proteins that take part in many cellular processes. The aim of this study was to show the influence of Trisenox (arsenic trioxide), on the cytoplasmic and nuclear F-actin organization. Arsenic trioxide is the proapoptotic factor. Together with increasing doses, it caused the increase in the number of cells undergoing apoptosis. Under arsenic trioxide treatment, cytoplasmic and nuclear F-actin (polymerized form of G-actin) was found reorganized. It was transformed into granulated structures. In cytometer studies fluorescence intensity of cytoplasmic F-actin after ATO treatment decreasing urgently in comparison to control. The obtained results may suggest the involvement of F-actin in apoptosis, especially in chromatin reorganization

Actin filament reorganization in HL-60 leukemia cell line after treatment with G-CSF and GM-CSF.

Currently, information regarding the influence of growth factors on the cytoskeleton, including G-CSF and GMCSF, remains limited. In the present study we show alterations in F-actin distribution and cell cycle progression in HL-60 promyelocytic leukemia cells, resulting from treatment with these cytokines in vitro. We found that both agents caused F-actin reorganization. Although multiple potential effects of various growth factors have been described previously, in our experimental conditions, we observed some rather subtle differences between the effects of G-CSF and GM-CSF on studied cells. The presence of these cytokines in the cell environment caused not only increased F-actin labeling in the cytoplasm, but also a weaker intensity of peripheral ring staining in comparison with control cells. In spite of the fact that HL60 cells exposed to G-CSF and GM-CSF contained different F-actin structures such as aggregates and F-actin network, the rate of actin polymerization was not significantly enhanced. Moreover, alterations were mainly related to considerable changes in the relative proportion of these different structures, what might be reflected by specific features of the differentiation process, with regard to the kind of stimulating factor used. Thus, reorganization of F-actin and other results obtained in our experimental conditions, might reflect unique characteristics of the differentiation process in HL-60 cells, involving low apoptosis frequency, the G1 to S phase transition in the cell cycle, as well as possible alternative ways of the cell death

The interactions between SATB1 and F-actin are important for mechanisms of active cell death

Introduction. The direct involvement of nuclear actin filaments in gene transcription and remodeling of chromatin is still debatable. However, nuclear localization of F-actin and its interactions with other nuclear matrix proteins have been reported. The aim of the study was to estimate the interactions between nuclear F-actin and one of the matrix proteins, special AT-rich sequence-binding protein 1 (SATB1), during active cell death induced in vitro by geldanamycin (GA). Material and methods. The expression of SATB1 was modified by the transfection of non-aggressive breast cancer MCF-7 cells with siRNA against SATB1 or expression plasmid with cloned cDNA of SATB1. The amount and localization of F-actin were altered by changes of cofilin-1 (CFL1) expression in MCF-7 cells. The association between SATB1 and F-actin during GA-induced cell death was analyzed using confocal and transmission electron microscopy. Results. Our studies revealed the colocalization between nuclear F-actin and SATB1 protein, during GA-induced death of breast cancer MCF-7 cells. The colocalization was enhanced in cells with overexpressed SATB1 and cofilin-1. At the ultrastructural level the SATB1 and F-actin complexes were seen at the border of condensed and decondensed chromatin. The presence of SATB1/F-actin molecular complexes was confirmed by magnetic separation of F-actin and interacting proteins. Conclusion. We suggest that the molecular interactions between SATB1 and F-actin are necessary for active cell death to occur

Analysis of serum homocysteine in the laboratory practice - comparison of the direct chemiluminescence immunoassay and high performance liquid chromatography coupled with fluorescent detection

Introduction: Effective diagnosis of cardiovascular diseases requires the right tools to be used enabling selective and sensitive analysis of their biomarkers. One of them is homocysteine (Hcy), nowadays determined by immunoassays and chromatographic methods. This study aims to compare the results obtained by direct chemiluminescence immunoassay (CLIA) and high performance liquid chromatography with fluorescent detection (HPLC-FD) using commercial kits. Materials and methods: Homocysteine concentration was determined in serum samples obtained from 101 individuals, using Atellica IM HCY (Siemens Healthineers, Erlangen, Germany) and HCY in plasma/serum – HPLC-FD (Chromsystems Instruments & Chemicals GmbH, Gräfelfing, Germany) tests validated for routine analysis. The latter was applied as a reference method. The comparability and agreement between the tested methods were evaluated using the Passing-Bablok (PB) regression analysis and the Bland-Altman (BA) method of the differences analysis. Results: Studies showed that CLIA gives higher Hcy concentrations (15.7 ± 4.14 μmol/L). Passing-Bablok regression analysis of the results obtained with CLIA (y) compared with HPLC-FD (x) yielded an intercept of 0.22 (95%CI: - 2.16 to 2.46) and slope of 1.58 (95%CI: 1.33 to 1.87). Bland-Altman analysis demonstrated a systematic positive bias for CLIA of 5.85 ± 2.77 μmol/L. Conclusions: Methods disagreement precludes their interchangeability. Lower Hcy values by HPLC-FD result from its greater selectivity. High performance liquid chromatography with fluorescent detection should be considered as preferential method for analysing Hcy in blood serum as well as the recommended reference method for routine clinical analysis. This fact, however, imposes the need to establish new reference ranges

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

 The cytoskeleton as an intracellular system plays an important role in the proper functioning of the cell. F-actin is one of the components, which built this structure. Microfilaments are involved in cell shape maintenance, polarity and cell motility. The aim of the present study was to determine the effect of doxorubicin on the actin reorganization and type of induced cell death in A549 cell line. In order to examine F-actin, the material was evaluated by the confocal and classical fluorescence microscope. Furthermore, changes in morphology and ultrastructure were analyzed by a light and transmission electron microscopy. The obtained data showed that doxorubicin causes a dosedependent decrease in A549 cell viability. Moreover, the treatment with doxorubicin resulted in the reorganization of F-actin as well as the induction of apoptosis and mitotic catastrophe in the non-small lung cancer cells. In addition, the existence of actin in the nucleus was confirmed.Cytoszkielet stanowi międzykomórkowy system, odgrywający istotną rolę w prawidłowym funkcjonowaniu każdej komórki. Jednym z jego komponentów jest F-aktyna, zaangażowana w zmiany kształtu, polarność, a także ruch komórek. Celem przedstawionej pracy było określenie wpływu doksorubicyny na reorganizację cytoszkieletu aktynowego oraz rodzaj indukowanej śmierci w komórkach linii A549. Wyniki badań oceniano przy użyciu klasycznego oraz konfokalnego mikroskopu fluorescencyjnego, a także z wykorzystaniem mikroskopii świetlnej oraz transmisyjnej mikroskopii elektronowej. W toku badań wykazano dawkozależną wrażliwość komórek linii A549 na doksorubicynę. Wraz ze wzrostem cytostatyku, wzrastał odsetek komórek martwych. Ponadto stwierdzono, że doksorubicyna powoduje zmiany w reorganizacji F-aktyny w komórkach niedrobnokomórkowego raka płuca, a także może indukować apoptozę oraz katastrofę mitotyczną. Dodatkowo potwierdzono występowanie aktyny na terenie jądra komórkowego

Possibilities in the application of solid lipid nanoparticles in combination with 5-fluorouracil to overcome the drugresistance of non-small cell lung cancer cell line A549

Introduction: Multidrug resistance of non-small cell lung cancer cells is associated with a high percentageof therapeutic failures. The aim of this study was to assess the ability of solid lipid nanoparticles as atransporter of the conventionally used cytostatic (5-fluorouracil) to overcome the resistance of A549 cells.Material and methods: MTT assay was used to assess the differences in viability of cells treated with5-fluorouracil alone or in combination with different types of solid lipid nanoparticles. Type of cell deathand distribution of cell cycle phases were evaluated using flow cytometry.Results: The use of nanoparticles as a 5-fluorouracil transporter reduced the viability of A549 cells to agreater extent than the cytostatic alone. This was mainly due to the increase in apoptosis, but also necrosisand cell cycle arrest.Conclusion: Our results indicate the great potential of nanotechnology in the treatment of non-small celllung cancer. By using nanoparticles, it is possible to sensitise tumour cells to cytostatics to which theyare normally resistant. In addition, literature data confirm the safety of solid lipid nanoparticle application