Maciej Gagat

Paclitaxel and the dietary flavonoid fisetin: a synergistic combination that induces mitotic catastrophe and autophagic cell death in A549 non-small cell lung cancer cells

AbstractBackground The use of the dietary polyphenols as chemosensitizing agents to enhance the efficacy of conventional cytostatic drugs has recently gained the attention of scientists and clinicians as a plausible approach for overcoming the limitations of chemotherapy (e.g. drug resistance and cytotoxicity). The aim of this study was to investigate whether a naturally occurring diet-based flavonoid, fisetin, at physiologically attainable concentrations, could act synergistically with clinically achievable doses of paclitaxel to produce growth inhibitory and/or pro-death effects on A549 non-small cell lung cancer cells, and if it does, what mechanisms might be involved.MethodsThe drug–drug interactions were analyzed based on the combination index method of Chou and Talalay and the data from MTT assays. To provide some insights into the mechanism underlying the synergistic action of fisetin and paclitaxel, selected morphological, biochemical and molecular parameters were examined, including the morphology of cell nuclei and mitotic spindles, the pattern of LC3-II immunostaining, the formation of autophagic vacuoles at the electron and fluorescence microscopic level, the disruption of cell membrane asymmetry/integrity, cell cycle progression and the expression level of LC3-II, Bax, Bcl-2 and caspase-3 mRNA.ResultsHere, we reported the first experimental evidence for the existence of synergism between fisetin and paclitaxel in the in vitro model of non-small cell lung cancer. This synergism was, at least partially, ascribed to the induction of mitotic catastrophe. The switch from the cytoprotective autophagy to the autophagic cell death was also implicated in the mechanism of the synergistic action of fisetin and paclitaxel in the A549 cells. In addition, we revealed that the synergism between fisetin and paclitaxel was cell line-specific as well as that fisetin synergizes with arsenic trioxide, but not with mitoxantrone and methotrexate in the A549 cells.ConclusionsOur results provide rationale for further testing of fisetin in the combination with paclitaxel or arsenic trioxide to obtain detailed insights into the mechanism of their synergistic action as well as to evaluate their toxicity towards normal cells in an animal model in vivo. We conclude that this study is potentially interesting for the development of novel chemotherapeutic approach to non-small cell lung cancer.

Hair follicle stem cells can be driven into a urothelial-like phenotype: an experimental study.

The aim of this study was to show that conditioned medium might induce transdifferentiation of hair follicle stem cells into urothelial‐like cells. Several conditioned media and culture conditions (skeletal muscle cell conditioned medium, smooth muscle cell conditioned medium, fibroblast conditioned medium, transforming growth factor‐conditioned medium, urothelial cell conditioned medium, and co‐culture of hair follicle stem cells and urothelial cells) were used. The hair follicle stem cells phenotype from rat whisker hair follicles was checked by using flow cytometry and immunofluorescence. Cytokeratins 7, 8, 15 and 18 were used as markers. Urothelial cell conditioned medium increased the expression of urothelial markers (cytokeratin 7, cytokeratin 8, cytokeratin 18), whereas it decreased a hair follicle stem cells marker (cytokeratin 15) after 2 weeks of culture. This process depended on the time of cultivation. This medium was able to sustain the epithelial phenotype of the culture. Other media including a co‐culture system failed to induce similar changes. Smooth muscle conditioned medium resulted in a loss of cells in culture. Hair follicle stem cells are capable of differentiating into urothelial‐like cells in vitro when exposed to a bladder‐specific microenvironment.

Actin is required for cellular death

Actin is one of the most abundant cytoskeletal proteins, which takes part in many cellular processes. This review provides information on the history, forms and localization of actin and its role, in particular in cellular death processes. We discuss the relationships between reorganization of actin filaments and apoptosis, mitotic catastrophe and differentiation. Finally, we discuss the translocation and accumulation of actin in the nuclear area. Moreover, owing to the difficulties of F-actin localization by transmission electron microscopy (TEM), the phalloidin-based method of its detection using streptavidin-coated quantum dots is presented in this review.

Doxorubicin-induced F-actin reorganization in cofilin-1 (nonmuscle) down-regulated CHO AA8 cells

The actin cytoskeleton plays an important role in many cellular processes, including cell mortality, mitosis, cytokinesis, intracellular transport, endocytosis and secretion but also is involved in gene transcription. The dynamics of the actin cytoskeleton is controlled by different classes of actin-binding proteins (ABPs) which regulate the polymerization of actin filaments. In this report we used siRNA against cofilin-1 (nonmuscle) to demonstrate the effect of cofilin on the nuclear and cytoplasmic actin pools in CHO AA8 cells after exposition to various concentrations of doxorubicin. The immunofluorescence studies showed doxorubicin dose dependent tendency to formation the multinucleated giant cells, but also the increase of fluorescence intensity of cofilin in nuclei of untransfected cells. Induction of cell death with doxorubicin treatment in untransfected cells revealed both mitotic catastrophe (in both lower and higher doxorubicin doses) and apoptosis (mostly in higher doxorubicin doses), whereas among cofilin-1 down-regulated cells we observed only mitotic catastrophe. The results suggest that cofilin has apoptosis-inducing ability and that mitotic catastrophe is independent from F-actin content in cell nucleus. In this point of view we conclude that different mechanisms of chromatin reorganization are involved in these two processes. Moreover, we suppose that apoptosis and mitotic catastrophe are independent from each other.

Ultrastructural localization of F-actin using phalloidin and quantum dots in HL-60 promyelocytic leukemia cell line after cell death induction by arsenic trioxide

Quantum dots (QDs) are fluorescent nanocrystals whose unique properties are fundamentally different from organic fluorophores. Moreover, their cores display sufficient electron density to be visible under transmission electron microscopy (TEM). Here, we report a technique for phalloidin-based TEM detection of F-actin. The ultrastructural reorganization of F-actin after arsenic trioxide (ATO) treatment was estimated using a combination of pre- and post-embedding techniques with biotinylated phalloidin and QD-streptavidin conjugates or colloidal gold (AU) conjugated to streptavidin. Ultrastructural studies showed ATO-induced apoptosis of HL-60 cells. Moreover, different patterns of QD-labeled F-actin after ATO treatment were seen. In the case of AU labeling, only a few gold particles were seen and it was impossible to see any difference in F-actin distribution. TEM imaging experiments using QDs and colloidal gold (AU) showed that the strategy of bioconjugation of nanoprobes is the most important factor in biotinylated phalloidin detection of F-actin using streptavidin-coated nanoparticles, especially at the ultrastructural level. Additionally, the results presented in present study confirm the essential role of F-actin in chromatin reorganization during cell death processes.

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.

Stem cells for urinary tract regeneration.

Regeneration of the urinary bladder is a complicated task, due to organ dimensions and diseases (cancer, interstitial cystitis) when autologous bladder cells cannot be used. Cancer is the most frequent indication for bladder removal (cystectomy). Stem cells can be used with the guarantee of the sufficient cell number for the in vitro construction of the urinary bladder wall. Tissue engineering techniques hold great promise for regeneration of dysfunctional urinary sphincter. Denervation following surgical procedures or injuries results in weakness of the urethral sphincter and stress urinary incontinence. Injectable therapies and the potential of stem cells for sphincter restoration was presented in this review. The aim of this review was to present possibilities of urinary bladder regeneration with the use of stem cells and tissue engineering techniques.

Filling Effects, Persistence, and Safety of Dermal Fillers Formulated With Stem Cells in an Animal Model

BACKGROUND Research is scarce regarding the effectiveness of dermal fillers containing autologous stem cells. OBJECTIVES The authors sought to determine the local and systemic effects of adipose-derived stem cells (ADSCs) as a component of dermal fillers in an animal model. METHODS Wistar rats were injected with 1 of the following dermal fillers: ADSCs combined with hyaluronic acid (ADSC-HA), ADSCs combined with fish collagen (ADSC-COL), HA alone (CONTROL-HA), or COL alone (CONTROL-COL). Fillers were injected into the glabella, dorsum, and chest of each animal. The ADSCs were labeled with PKH26 to assess cell migration. Filling effects (FEs) were measured immediately after injection and at 1.5 months and 3 months after injection. Skin specimens were stained with hematoxylin and eosin to assess localization and persistence of ADSCs. RESULTS Mean FEs in animals implanted with ADSCs were greater and persisted longer than those of controls. No inflammatory responses were observed in any group. Three months after injection, PKH26-positive cells comprised nearly 70% of cells at the injection site in animals treated with ADSC-HA. PKH26 fluorescence also was detected in the spleen but not in the brain, kidney, or lung. CONCLUSIONS Stem cells have the potential to improve the aesthetic effects and longevity of dermal fillers.

Tropomyosin-1 protects endothelial cell-cell junctions against cigarette smoke extract through F-actin stabilization in EA.hy926 cell line.

The aim of the study was to estimate the effect of cigarette smoke extract (CSE) on EA.hy926 endothelial cells in culture in the context of maintenance of cell-cell junctions through the structural stabilization of the actin cytoskeleton. In the present study, F-actin was stabilized by the overexpression of tropomyosin-1, which is known to stabilize actin filaments in muscle and non-muscle cells. Our study showed that the stabilization of F-actin significantly increased the survival of cells treated with 25% CSE. In addition, after stabilization of F-actin the migratory potential of EA.hy926 cells subjected to CSE treatment was increased. Our results also showed increased fluorescence intensity of alpha- and beta-catenin after CSE treatment in cells which had stabilized F-actin. Analysis of fluorescence intensity of Zonula occludens-1 did not reveal any significant differences when EA.hy926 cells overexpressing tropomyosin-1 were compared with those lacking overexpression. It would appear that overexpression of tropomyosin-1 preserved the structure of actin filaments in the cells treated with CSE. In conclusion, the present study demonstrates that stabilization of F-actin protects EA.hy926 cells against CSE-induced loss of both adherens and tight junctions. The data presented in this study suggest that overexpression of tropomyosin-1 stabilizes the organizational structure of actin filaments and helps preserve the endothelial barrier function under conditions of strong oxidative stress.

Involvement of the SATB1/F-actin complex in chromatin reorganization during active cell death

Over the past years, confirmations on the presence of actin and/or its polymerized form, F-actin, in the cell nucleus are progressively accumulating. Nevertheless, the function and localization of F-actin in the nucleus is still not fully characterized. Thus, the aim of the present study was to evaluate the association between F-actin and sequence-binding protein 1 (SATB1) and their involvement in chromatin remodeling associated with active cell death. Both SATB1 and F-actin were colocalized in the transcriptional active regions of the cell nucleus and a functional interaction was observed between SATB1 and higher-organized nuclear F-actin structures at the border between condensed and decondensed chromatin. These results extend the knowledge on the role of SATB1 and nuclear F-actin in three-dimensional chromatin organization and their functions during active cell death. Additionally, this study opens the discussion on the involvement of the SATB1/F-actin functional complex in active cell death; further studies are required to fully elucidate these issues.