Actin Cytoskeleton Remodeling Accompanied by Redistribution of Adhesion Proteins Drives Migration of Cells in Different EMT States.

Epithelial-mesenchymal transition (EMT) is a process during which epithelial cells lose epithelial characteristics and gain mesenchymal features. Here, we used several cell models to study migratory activity and redistribution of cell-cell adhesion proteins in cells in different EMT states: EGF-induced EMT of epithelial IAR-20 cells; IAR-6-1 cells with a hybrid epithelial-mesenchymal phenotype; and their more mesenchymal derivatives, IAR-6-1-DNE cells lacking adherens junctions. In migrating cells, the cell-cell adhesion protein α-catenin accumulated at the leading edges along with ArpC2/p34 and α-actinin. Suppression of α-catenin shifted cell morphology from fibroblast-like to discoid and attenuated cell migration. Expression of exogenous α-catenin in MDA-MB-468 cells devoid of α-catenin drastically increased their migratory capabilities. The Y654 phosphorylated form of ß-catenin was detected at integrin adhesion complexes (IACs). Co-immunoprecipitation studies indicated that α-catenin and pY654-ß-catenin were associated with IAC proteins: vinculin, zyxin, and α-actinin. Taken together, these data suggest that in cells undergoing EMT, catenins not participating in assembly of adherens junctions may affect cell migration.

X-ray and UV Irradiation-Induced Reactive Oxygen Species Mediated Antibacterial Activity in Fe and Pt Nanoparticle-Decorated Si-Doped TiCaCON Films.

Bone implants with biocompatibility and the ability to biomineralize and suppress infection are in high demand. The occurrence of early infections after implant placement often leads to repeated surgical treatment due to the ineffectiveness of antibiotic therapy. Therefore, an extremely attractive solution to this problem would be the ability to initiate bacterial protection of the implant by an external influence. Here, we present a proof-of-concept study based on the generation of reactive oxygen species (ROS) by the implant surface in response to X-ray irradiation, including through a layer of 3 mm adipose tissue, providing bactericidal protection. The effect of UV and X-ray irradiation of the implant surface on the ROS formation and the associated bactericidal activity was compared. The focus of our study was light-sensitive Si-doped TiCaCON films decorated with Fe and Pt nanoparticles (NPs) with photoinduced antibacterial activity mediated by ROS. In the visible and infrared range of 300-1600 nm, the films absorb more than 60% of the incident light. The high light absorption capacity of TiO2/TiC and TiO2/TiN heterostructures was demonstrated by density functional theory calculations. After short-term (5-10 s) low-dose X-ray irradiation, the films generated significantly more ROS than after UV illumination for 1 h. The Fe/TiCaCON-Si films showed enhanced biomineralization capacity, superior cytocompatibility, and excellent antibacterial activity against multidrug-resistant hospital Escherichia coli U20 and K261 strains and methicillin-resistant Staphylococcus aureus MW2 strain. Our study clearly demonstrates that oxidized Fe NPs are a promising alternative to the widely used Ag NPs in antibacterial coatings, and X-rays can potentially be used in ROS-regulating therapy to suppress inflammation in case of postimplant complications.

Epithelial-Mesenchymal Transition of Breast Cancer Cells Induced by Activation of the Transcription Factor Snail1.

Cancer cells use the program of epithelial-mesenchymal transition (EMT) for initiation of the invasion-metastasis cascade. Using confocal and video-microscopy, reorganization of the cytoskeleton was studied in the MCF-7 breast cancer cells undergoing Snail1-induced EMT. We used the line of MCF-7 cells stably expressing tetOff SNAI1 construct (MCF-7-SNAI1 cells). After tetracycline washout and Snail1 activation MCF-7-SNAI1 cells underwent EMT and acquired a migratory phenotype while retaining expression of E-cadherin. We identified five variants of the mesenchymal phenotype, differing in cell morphology and migration velocity. Migrating cells had high degree of plasticity, which allowed them to quickly change both the phenotype and migration velocity. The changes of the phenotype of MCF-7-SNAI1 cells are based on the Arp2/3-mediated branched actin network polymerization in lamellipodia, myosin-based contractility in the zone behind the nucleus, redistribution of adhesive proteins from cell-cell contacts to the leading edge, and reorganization of intermediate keratin filaments.

Dual role of E-cadherin in cancer cells.

E-cadherin is the main component of epithelial adherens junctions (AJs), which play a crucial role in the maintenance of stable cell-cell adhesion and overall tissue integrity. Down-regulation of E-cadherin expression has been found in many carcinomas, and loss of E-cadherin is generally associated with poor prognosis in patients. During the last decade, however, numerous studies have shown that E-cadherin is essential for several aspects of cancer cell biology that contribute to cancer progression, most importantly, active cell migration. In this review, we summarize the available data about the input of E-cadherin in cancer progression, focusing on the latest advances in the research of the various roles E-cadherin-based AJs play in cancer cell dissemination. The review also touches upon the "cadherin switching" in cancer cells where N- or P-cadherin replace or are co-expressed with E-cadherin and its influence on the migratory properties of cancer cells.

Phenotypic Plasticity of Cancer Cells Based on Remodeling of the Actin Cytoskeleton and Adhesive Structures.

There is ample evidence that, instead of a binary switch, epithelial-mesenchymal transition (EMT) in cancer results in a flexible array of phenotypes, each one uniquely suited to a stage in the invasion-metastasis cascade. The phenotypic plasticity of epithelium-derived cancer cells gives them an edge in surviving and thriving in alien environments. This review describes in detail the actin cytoskeleton and E-cadherin-based adherens junction rearrangements that cancer cells need to implement in order to achieve the advantageous epithelial/mesenchymal phenotype and plasticity of migratory phenotypes that can arise from partial EMT.

Early Events in Actin Cytoskeleton Dynamics and E-Cadherin-Mediated Cell-Cell Adhesion during Epithelial-Mesenchymal Transition.

Epithelial-mesenchymal transition (EMT) plays an important role in development and also in initiation of metastasis during cancer. Disruption of cell-cell contacts during EMT allowing cells to detach from and migrate away from their neighbors remains poorly understood. Using immunofluorescent staining and live-cell imaging, we analyzed early events during EMT induced by epidermal growth factor (EGF) in IAR-20 normal epithelial cells. Control cells demonstrated stable adherens junctions (AJs) and robust contact paralysis, whereas addition of EGF caused rapid dynamic changes at the cell-cell boundaries: fragmentation of the circumferential actin bundle, assembly of actin network in lamellipodia, and retrograde flow. Simultaneously, an actin-binding protein EPLIN was phosphorylated, which may have decreased the stability of the circumferential actin bundle. Addition of EGF caused gradual replacement of linear E-cadherin-based AJs with dynamic and unstable punctate AJs, which, unlike linear AJs, colocalized with the mechanosensitive protein zyxin, confirming generation of centripetal force at the sites of cell-cell contacts during EMT. Our data show that early EMT promotes heightened dynamics at the cell-cell boundaries-replacement of stable AJs and actin structures with dynamic ones-which results in overall weakening of cell-cell adhesion, thus priming the cells for front-rear polarization and eventual migration.

An In Vitro System to Study the Epithelial-Mesenchymal Transition In Vitro.

The epithelial-mesenchymal transition (EMT) plays an important role in development and cancer progression. Upon EMT, epithelial cells lose stable cell-cell adhesions and reorganize their cytoskeleton to acquire migratory activity. Recent data demonstrated that EMT drives cancer cells from the epithelial state to a hybrid epithelial/mesenchymal phenotype with retention of some epithelial markers (in particular, E-cadherin), which is important for cancer cell dissemination. In vitro studies of the effect of growth factors (in particular, epidermal growth factor (EGF)) on cultured cells can be highly advantageous for understanding the details of the early stages of EMT. The methods described in this chapter are intended for studying intermediate phenotypes of EMT. Time-lapse DIC microscopy is used for visualization of changes in morphology and motility of the cells stimulated with EGF. The transwell migration assay allows the evaluation of the migratory activity of the cells. Studying of dynamics of a fluorescently labeled actin-binding protein F-tractin-tdTomato using confocal microscopy allows detection of EGF-induced changes in the organization of the actin cytoskeleton. Live-cell imaging of cells stably expressing GFP-E-cadherin visualizes reorganization of stable tangential E-cadherin-based adherens junctions (AJs) into unstable radial AJs during the early stages of EMT.

Effect of BN Nanoparticles Loaded with Doxorubicin on Tumor Cells with Multiple Drug Resistance.

Herein we study the effect of doxorubicin-loaded BN nanoparticles (DOX-BNNPs) on cell lines that differ in the multidrug resistance (MDR), namely KB-3-1 and MDR KB-8-5 cervical carcinoma lines, and K562 and MDR i-S9 leukemia lines. We aim at revealing the possible differences in the cytotoxic effect of free DOX and DOX-BNNP nanoconjugates on these types of cells. The spectrophotometric measurements have demonstrated that the maximum amount of DOX in the DOX-BNNPs is obtained after saturation in alkaline solution (pH 8.4), indicating the high efficiency of BNNPs saturation with DOX. DOX release from DOX-BNNPs is a pH-dependent and DOX is more effectively released in acid medium (pH 4.0-5.0). Confocal laser scanning microscopy has shown that the DOX-BNNPs are internalized by neoplastic cells using endocytic pathway and distributed in cell cytoplasm near the nucleus. The cytotoxic studies have demonstrated a higher sensitivity of the leukemia lines to DOX-BNNPs compared with the carcinoma lines: IC50(DOX-BNNPs) is 1.13, 4.68, 0.025, and 0.14 µg/mL for the KB-3-1, MDR KB-8-5, K562, and MDR i-S9 cell lines, respectively. To uncover the mechanism of cytotoxic effect of nanocarriers on MDR cells, DOX distribution in both the nucleus and cytoplasm has been studied. The results indicate that the DOX-BNNP nanoconjugates significantly change the dynamics of DOX accumulation in the nuclei of both KB-3-1 and KB-8-5 cells. Unlike free DOX, the utilization of DOX-BNNPs nanoconjugates allows for maintaining a high and stable level of DOX in the nucleus of MDR KB-8-5 cells.

Cadherin-mediated cell-cell interactions in normal and cancer cells.

Adherens junctions (AJs) are molecular complexes that mediate cell-cell adhesive interactions and play pivotal roles in maintenance of tissue organization in adult organisms and at various stages of development. AJs consist of cadherin adhesion receptors, providing homophilic ligation with cadherins on adjacent cells, and members of the catenin protein family: p120, ß- and α-catenin. α-catenin's linkage with the actin cytoskeleton defines the linear or punctate organization of AJs in different cell types. Myosin II-dependent tension drives vinculin recruitment by α-catenin and stabilizes the linkage of the cadherin/catenin complex to F-actin. Neoplastic transformation leads to prominent changes in the organization, regulation and stability of AJs. Epithelial-mesenchymal transition (EMT) whereby epithelial cells lose stable cell-cell adhesion, and reorganize their cytoskeleton to acquire migratory activity, plays the central role in cancer cell invasion and metastasis. Recent data demonstrated that a partial EMT resulting in a hybrid epithelial/mesenchymal phenotype with retention of E-cadherin is essential for cancer cell dissemination. E-cadherin and E-cadherin-based AJs are required for collective invasion and migration, survival in circulation, and metastatic outgrowth.

A Novel Role of E-Cadherin-Based Adherens Junctions in Neoplastic Cell Dissemination.

Using confocal microscopy, we analyzed the behavior of IAR-6-1, IAR1170, and IAR1162 transformed epithelial cells seeded onto the confluent monolayer of normal IAR-2 epithelial cells. Live-cell imaging of neoplastic cells stably expressing EGFP and of normal epithelial cells stably expressing mKate2 showed that transformed cells retaining expression of E-cadherin were able to migrate over the IAR-2 epithelial monolayer and invade the monolayer. Transformed IAR cells invaded the IAR-2 monolayer at the boundaries between normal cells. Studying interactions of IAR-6-1 transformed cells stably expressing GFP-E-cadherin with the IAR-2 epithelial monolayer, we found that IAR-6-1 cells established E-cadherin-based adhesions with normal epithelial cells: dot-like dynamic E-cadherin-based adhesions in protrusions and large adherens junctions at the cell sides and rear. A comparative study of a panel of transformed IAR cells that differ by their ability to form E-cadherin-based AJs, either through loss of E-cadherin expression or through expression of a dominant negative E-cadherin mutant, demonstrated that E-cadherin-based AJs are key mediators of the interactions between neoplastic and normal epithelial cells. IAR-6-1DNE cells expressing a dominant-negative mutant form of E-cadherin with the mutation in the first extracellular domain practically lost the ability to adhere to IAR-2 cells and invade the IAR-2 epithelial monolayer. The ability of cancer cells to form E-cadherin-based AJs with the surrounding normal epithelial cells may play an important role in driving cancer cell dissemination in the body.

Rearrangements of the actin cytoskeleton and E-cadherin-based adherens junctions caused by neoplasic transformation change cell-cell interactions.

E-cadherin-mediated cell-cell adhesion, which is essential for the maintenance of the architecture and integrity of epithelial tissues, is often lost during carcinoma progression. To better understand the nature of alterations of cell-cell interactions at the early stages of neoplastic evolution of epithelial cells, we examined the line of nontransformed IAR-2 epithelial cells and their descendants, lines of IAR-6-1 epithelial cells transformed with dimethylnitrosamine and IAR1170 cells transformed with N-RasG12D. IAR-6-1 and IAR1170 cells retained E-cadherin, displayed discoid or polygonal morphology, and formed monolayers similar to IAR-2 monolayer. Fluorescence staining, however, showed that in IAR1170 and IAR-6-1 cells the marginal actin bundle, which is typical of nontransformed IAR-2 cells, disappeared, and the continuous adhesion belt (tangential adherens junctions (AJs)) was replaced by radially oriented E-cadherin-based AJs. Time-lapse imaging of IAR-6-1 cells stably transfected with GFP-E-cadherin revealed that AJs in transformed cells are very dynamic and unstable. The regulation of AJ assembly by Rho family small GTPases was different in nontransformed and in transformed IAR epithelial cells. As our experiments with the ROCK inhibitor Y-27632 and the myosin II inhibitor blebbistatin have shown, the formation and maintenance of radial AJs critically depend on myosin II-mediated contractility. Using the RNAi technique for the depletion of mDia1 and loading cells with N17Rac, we established that mDia1 and Rac are involved in the assembly of tangential AJs in nontransformed epithelial cells but not in radial AJs in transformed cells. Neoplastic transformation changed cell-cell interactions, preventing contact paralysis after the establishment of cell-cell contact and promoting dynamic cell-cell adhesion and motile behavior of cells. It is suggested that the disappearance of the marginal actin bundle and rearrangements of AJs may change the adhesive function of E-cadherin and play an active role in migratory activity of carcinoma cells.