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 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

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

Guidelines of the Polish Respiratory Society for diagnosis and treatment of idiopathic pulmonary fibrosis

Introduction: This document presents the guidelines of the Polish Respiratory Society (PTChP, Polskie Towarzystwo Chorób Płuc) for diagnosis and treatment of idiopathic pulmonary fibrosis (IPF), developed by a group of Polish experts.Material and methods: The recommendations were developed in the form of answers to previously formulated questions concer-ning everyday diagnostic and therapeutic challenges. They were developed based on a current literature review using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology.Results: We formulated 28 recommendations for diagnosis (8), pharmacological treatment (12) as well as non-pharma-cological and palliative therapy (8). The experts suggest that surgical lung biopsy (SLB) not be performed in patients with the probable usual interstitial pneumonia (UIP) pattern, with an appropriate clinical context and unanimous opinion of a  multidisciplinary team. The experts recommend using antifibrotic agents in IPF patients and suggest their use irrespective of the degree of functional impairment. As regards non-pharmacological and palliative treatment, strong re-commendations were formulated regarding pulmonary rehabilitation, oxygen therapy (in patients with chronic respiratory failure), preventive vaccinations as well as referring IPF patients to transplant centres. Table 1 presents an aggregate list of recommendations.Conclusions: The Polish Respiratory Society Working Group developed guidelines for IPF diagnosis and treatment

SARS-CoV-2 infects an <I>in vitro</I> model of the human developing pancreas through endocytosis

Recent studies showed that SARS-CoV-2 can infect adult human pancreas and trigger pancreatic damage. Here, using human fetal pancreas samples and 3D differentiation of human pluripotent cells into pancreatic endocrine cells, we determined that SARS-CoV-2 receptors ACE2, TMPRSS2, and NRP1 are expressed in precursors of insulin-producing pancreatic β-cells, rendering them permissive to SARS-CoV-2 infection. We also show that SARS-CoV-2 enters and undergoes efficient replication in human multipotent pancreatic and endocrine progenitors in vitro. Moreover, we investigated mechanisms by which SARS-CoV-2 enters pancreatic cells, and found that ACE2 mediates the entry, while NRP1 and TMPRSS2 do not. Surprisingly, we found that in pancreatic progenitors, SARS-CoV-2 enters cells via cathepsin-dependent endocytosis, which is a different route than in respiratory tract. Therefore, pancreatic spheroids might serve as a model to study candidate drugs for endocytosis-mediated viral entry inhibition and to investigate whether SARS-CoV-2 infection may affect pancreas development, possibly causing lifelong health consequences

HA PEGylated Filler in Association with an Infrared Energy Device for the Treatment of Facial Skin Aging: 150 Day Follow-Up Data Report

Background: The face is the area most exposed to the normal course of skin aging, both intrinsically and extrinsically. The aim of the study was to evaluate the cellular and clinical response of a therapeutic protocol aimed at countering facial skin aging. Materials and methods: Twenty female patients with facial skin laxity and photodamage underwent combined therapy including mesotherapy using non-cross-linked hyaluronic acid with calcium hydroxyapatite and an infrared energy-based device treatment with subsequent implementation of PEG-cross-linked hyaluronic acid soft tissue fillers. To evaluate the benefits, patients underwent histological, immunological, and biomechanical evaluations before the treatment and at 21 and 150 days after the treatment. Results: The histological results at 21 days and 150 days after the procedure showed an increase in the number of fibroblasts and angiogenesis. As for the immunological aspect, it was shown that the treatment has an immunomodulating action, avoiding the activation of CD4 and CD8 cells. Biomechanical data showed that, at 150 days after treatment, the average changes in skin elasticity increased by 72% and the skin hydration increased by 49%. Conclusions: A combination of an infrared energy-based device treatment with both non-cross-linked hyaluronic acid and novel PEG-cross-linked hyaluronic acid leads to numerous positive cutaneous changes after histological, immunological, and biomechanical evaluations

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