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

Neovascularization in Meniscus and Tendon Pathology as a Potential Mechanism in Regenerative Therapies: Special Reference to Platelet-Rich Plasma Treatment

Neovascularization is a complex, multistep process that includes the activation of endothelial cells, degradation of the basement membrane surrounding the blood vessel, formation of tip cells, the sprouting, migration and proliferation of endothelial cells into the interstitial space, and then the generation of space in the matrix to allow for the formation of a new, proper lumen of a newly formed blood vessel. Abundant neovascularization can be found in tendinous tissue obtained from asymptomatic athletes or the meniscus early after the injury. The concept of neovascularization in musculoskeletal system disorders seems to be mainly associated with pain and poor clinical outcomes. On the one hand, this phenomenon allows for tissue regeneration, but on the other, it is present during the degeneration process in connective tissue. Establishing the current concept on neovascularization is also needed. A narrative review of the current literature was conducted using databases including Embase, PubMed and Cochrane. This review aims to investigate the exact role of the neovascularization process in tendon and meniscus lesions and its role as a potential target in clinics, specifically in platelet-rich plasma (PRP) therapy. The stabilization of the neovessels required to achieve the healed tissue, together with the standardization of the PRP injections, can offer an alternative future therapeutic approach for the treatment of tendinopathy and meniscal injuries

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

Wpływ łagodnej hipertermii na morfologię, ultrastrukturę i organizację F- Aktyny w komórkach linii HL-60

Introduction. Hyperthermia is a well-established physical stimulus, which is applied as an adjunctive therapy with various cancer treatments, such as radiotherapy and chemotherapy. However, the precise mechanism of heat action at the cellular level remains to be elucidated, and appears to be multi-dimensional. The purpose of the current study was to determine the effect of mild hyperthermia on the actin cytoskeleton in the HL-60 cell line. In addition, the morphological and ultrastructural approaches were used to determine the type of hyperthermia-induced cell death.Material and methods. All studies were performed using human promyelocytic leukemia cell line (HL-60). Actin filaments were visualized with phalloidin conjugated to Alexa Fluor® 488 using fluorescence microscopy. Morphological and ultrastructural changes in the HL-60 cells were analysed by light and electron microscopy, respectively.Results. Exposure of HL-60 cells to mild hyperthermia resulted in the reorganization of the actin cytoskeleton and the appearance of characteristic apoptotic features, including cell shrinkage, chromatin condensation and margination. In addition, swollen mitochondria were observed. The morphological and ultrastructural changes increased in severity with an increase in recovery time. Similarly, actin filament remodeling was observed immediately after the heat shock and was more evident 3 and 6 hrs after the treatment. These effects were mainly reflected by a higher definition of the dense cortical F-actin ring as well as the appearance of brightly fluorescent F-actin dots and networks scattered throughout the cytoplasm.Conclusions. Presented data suggest that actin filament reorganization is involved in the process of apoptosis initiated by mild hyperthermia. Furthermore, the results of our studies showed that the severity of hyperthermia-induced morphological and ultrastructural changes as well as alterations in actin organization depend not only on the temperature treatment but also on the duration of post heat shock recoveryWstęp. Hipertermia jest dynamicznie rozwijającą się metodą lecznia nowotworów, stosowaną w skojarzeniu z radio- i/lub chemioterapią. Precyzyjny mechanizm działania hipertermii na poziomie komórkowym nie został, jak dotąd w pełni poznany i wydaje się on wielowymiarowy. Celem niniejszej pracy była analiza wpływu łagodnej hipertermii na organizację filamentów aktynowych w komórkach linii HL-60. Za zasadną uznano także ocenę zmian morfologicznych i ultrastrukturalnych wywołanych przez hipertermię, celem określenia rodzaju uruchamianej śmierci zachodzącej w badanej linii.Materiały i metody . Badania przeprowadzono na komórkach linii białaczki promielocytowej HL-60. Filameny aktynowe wyznakowano falloidyną skoniugowaną z Alexa Fluor® 488 i oglądano w mikroskopie fluorescencyjnym. Morfologiczne i ultrastrukturalne zmiany w komórkach oceniono odpowiednio, przy użyciu mikroskopu świetlnego oraz transmisyjnego mikroskopu elektronowego.Wyniki. W wyniku ekspozycji komórek HL-60 na podwyższoną temperaturę obserwowano reorganizację cytoszkieletu aktynowego oraz pojawienie się komórek o cechach charakterystycznych dla procesu apoptozy, takich jak obkurcznie cyto- i nukleoplazmy, kondensacja i marginalizacja chromatyny czy obrzmienie mitochondriów. W wyniku rearanżacji, F-aktyna lokalizowała sie głównie w części korowej komórki w formie pierścienia lub w cytoplazmie w postaci wyraźnie wyznakowanych sieci i skupień. Stopień nasilenia zmian w komórkach wzrastał wraz ze wzrostem czasu regeneracji komórek.Wnioski. Uzyskane wyniki pozwalają stwierdzić, że cytoszkielet aktynowy zaangażowany jest w realizację procesu apoptozy indukowanej przez łagodną hipertermię. Ponadto sugerują one, że na wystąpienie zmian w organizacji filamentów aktynowych, jak również zmian w morfologii i ultrastrukturze komórek ma wpływ, nie tylko zastosowany profil temperaturowy, ale również czas regeneracji komórek

Expression of cyclin B1 after induction of senescence and cell death in non-small cell lung carcinoma A549 cells

The purpose of this study was to evaluate the level of mitotic cyclin B1 in the context of senescence and cell death in A549 non-small cell lung carcinoma cells. This was performed through analysis of the cell cycle, the percentage of SA-β-galactosidase-positive, as well as TUNEL-positive cells. Morphological alterations were studied using a transmission electron microscope. Changes in  the intracellular level and the presence of cyclin B1 in the nucleus and cytoplasm areas were detected by flow cytometry and confocal fluorescence microscopy, respectively. In the cells exposed to various concentrations of doxorubicin, different kinds of cell death and senescent phenotype were observed. Alterations in the cell cycle and increased polyploidy may be indicative of mitotic catastrophe execution. Changes in cyclin B1 may also be strictly related to its different regulation at mitotic catastrophe and senescence programs

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

Can bone marrow mesenchymal stem cells regenerate the myocardium?

Background: Cardiovascular diseases are the serious clinical problem, especially the loss of viable myocytes.A new approach, which provides a novel method for the treatment, is a tissue engineering andregenerative medicine. One of the current cell types used as a source to improve cardiac tissue repair,are MSCs. The aim of this study was to check if the 5’azacitidine and growth factors from cardiomyocytecell line initiate the differentiation of MSCs toward cardiomyocytes. Material and methods: Bone marrow MSCs were isolated and their biological features have been characterized.Conditioned media were prepared with the use of 5’azacitidine and growth factors releasedfrom cardiomyocytes. Results: The transdifferentiating process has been confirmed by the expression of specific muscle markers.Conditioned medium from cardiomyocyte cell line, as well as, 5’azacitidine induced muscle differentiationprocess in bone marrow MSCs in comparison to the control. Conclusions: Presented in this study data support the conclusion that this concept may represent a promisingstrategy for the repair of cardiac tissue, however, further experiments are necessary

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

WPŁYW DOKSORUBICYNY NA JĄDROWĄ I CYTOPLAZMATYCZNĄ PULĘ F-AKTYNY W LINII KOMÓRKOWEJ A549

 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