THz irradiation inhibits cell division by affecting actin dynamics.

Biological phenomena induced by terahertz (THz) irradiation are described in recent reports, but underlying mechanisms, structural and dynamical change of specific molecules are still unclear. In this paper, we performed time-lapse morphological analysis of human cells and found that THz irradiation halts cell division at cytokinesis. At the end of cytokinesis, the contractile ring, which consists of filamentous actin (F-actin), needs to disappear; however, it remained for 1 hour under THz irradiation. Induction of the functional structures of F-actin was also observed in interphase cells. Similar phenomena were also observed under chemical treatment (jasplakinolide), indicating that THz irradiation assists actin polymerization. We previously reported that THz irradiation enhances the polymerization of purified actin in vitro; our current work shows that it increases cytoplasmic F-actin in vivo. Thus, we identified one of the key biomechanisms affected by THz waves.

Thymoquinone Enhances the Effect of Gamma Knife in B16-F10 Melanoma Through Inhibition of Phosphorylated STAT3.

BACKGROUND: Patients with brain metastasis from melanoma have a dismal prognosis with poor survival time. Gamma Knife (GK) is an effective treatment to control brain metastasis from melanoma. Thymoquinone (TQ) has emerged as a potential therapeutic option due to its antiproliferative effects on various cancers. The purpose of the study was to assess the effect of GK on B16-F10 melanoma cells in vitro and intracerebral melanoma in vivo, and its synergistic effect in combination with TQ. METHODS: The effects of GK and combination treatment of GK and TQ were studied on B16-F10 melanoma cells by evaluating cytotoxicity with an adenosine triphosphate assay, apoptosis by acridine orange staining, and genotoxicity by comet assay. Western blot analysis was performed to investigate the expression of STAT3, p-STAT3 (Tyr705), JAK2, p-JAK2, caspase-3, Bax, Bcl-2, survivin, and ß-actin. Expression of inflammatory cytokines was assessed by enzyme-linked immunosorbent assay. GK alone and in combination with TQ was assessed in an established intracerebral melanoma tumor in mice. RESULTS: The effects of GK on cytotoxicity, genotoxicity, and apoptosis were enhanced by TQ in B16-F10 melanoma cells. GK induced apoptosis through inhibition of p-STAT3 expression, which in turn regulated pro- and antiapoptotic proteins such as caspase-3, Bax, Bcl-2, and survivin. Adding TQ to GK irradiation further enhanced this apoptotic effect of GK irradiation. GK was shown to reduce the levels of tumor-related inflammatory cytokines in B16-F10 melanoma cells. This effect was more pronounced when TQ was added to GK irradiation. GK with 15 Gy increased the survival of mice with intracerebral melanoma compared with untreated mice. However, despite the additive effect of TQ in addition to GK irradiation on B16-F10 melanoma cells in vitro, TQ did not add any significant survival benefit to GK treatment in mice with intracerebral melanoma. CONCLUSIONS: Our findings suggest that TQ would be a potential therapeutic agent in addition to GK to enhance the antitumor effect of irradiation. Further studies are required to support our findings.

Effect of bronchial thermoplasty on structural changes and inflammatory mediators in the airways of subjects with severe asthma.

BACKGROUND: Bronchial thermoplasty (BT) is a novel technique used in the treatment of subjects with severe refractory asthma. Radiofrequency is provided to airway walls during bronchoscopy in order to reduce airway remodeling. Several clinical studies have reported an improvement in subjects' symptoms following BT. However, how BT affects the airway architectures and inflammatory mediators in the airways has not been yet fully elucidated. METHODS: Fourteen subjects with severe asthma were recruited in this study according to the criteria of ATS severe asthma definition. The study subjects undertook bronchial biopsy during the bronchoscopy procedure at baseline and 6 weeks after the initial BT treatment. The obtained samples were stained with antibodies for α-smooth muscle actin (α-SMA); protein gene product (PGP) 9.5, a specific nerve marker; von Willebrand factor (vWF), a marker for blood vessels; interleukin-17A (IL-17A) and transforming growth factor-ß1 (TGF-ß1). RESULTS: The expression of α-SMA and PGP9.5 were significantly reduced post-BT. There was no significant difference in the number of blood vessels between baseline and post-BT. In addition, BT did not affect the production of IL-17A and TGF-ß1 in the airways. The changes in the expression of α-SMA and PGP9.5 had no significant correlation with the improvement of pulmonary function. CONCLUSION: and Clinical Relevance: This study suggests that BT reduces airway smooth muscle mass and the airway innervation without affecting vasculature and the production of inflammatory mediators in the airways of subjects with severe asthma.

Actin polymerization is activated by terahertz irradiation.

Polymerization of monomeric actin into filaments has pivotal roles in cell motility, growth, differentiation, and gene expression. Therefore, techniques of manipulating actin polymerization, including actin-binding chemicals, have been developed for understanding and regulating multiple biological functions. Here, we demonstrate that irradiation with terahertz (THz) waves is a novel method of modulating actin polymerization. When actin polymerization reaction is performed under irradiation with 0.46 THz waves generated by a Gyrotron, actin polymerization was observed to be activated by monitoring the fluorescence of pyrene actin fluorophores. We also observed the number of actin filaments under a fluorescence microscope using the polymerized actin probe SiR-actin. The number of actin filaments was increased by 3.5-fold after THz irradiation for 20 min. When the THz irradiation was applied to a steady-state actin solution, in which elongation and depolymerization of actin filaments were equilibrated, increased actin polymerization was observed, suggesting that the THz irradiation activates actin polymerization, at least in the elongation process. These results suggest that THz waves could be applied for manipulating biomolecules and cells.

Photoactivation of Actin in Mouse Oocyte.

Development of fluorescence distribution assays like FRAP (fluorescence recovery after photobleaching) or photoactivation has had a great impact in studying intracellular protein dynamics. In particular, the cytoskeleton field largely benefited from these techniques, with lots of new information provided about the dynamics and organization of actin networks whithin cells.In mouse oocyte, actin photoactivation has been very useful to determine the dynamics of different actin structures involved in meiotic divisions, including a cytoplasmic meshwork and a subcortical actin layer.Here, we describe a method, actin photoactivation, to determine the dynamics of the actin cytoplasmic meshwork and the subcortical actin layer during the first meiotic division in the mouse oocyte, that could be adapted to other actin structures or other stages of meiotic divisions.

Impact of neoadjuvant therapy on cancer-associated fibroblasts in rectal cancer.

BACKGROUND AND PURPOSE: Cancer-associated fibroblasts (CAFs) are increasingly recognised as promoters of tumour progression. It is poorly investigated whether cancer management protocols, such as neoadjuvant radio(chemo)therapy, have an impact on CAFs and, by consequence, on tumour progression. This prompted us to study the impact of neoadjuvant radio(chemo)therapy on the α-SMA/epithelial area ratio in rectal cancer, and the impact of this ratio on recurrence-free survival. MATERIAL AND METHODS: Immunohistochemistry for the CAF marker α-SMA and the proliferation marker Ki67 was performed on sections from 98 rectal cancers of which 62 had undergone neoadjuvant radio(chemo)therapy. RESULTS: Computer-assisted quantitative analysis showed that the α-SMA/neoplastic epithelial area ratio was higher after neoadjuvant therapy, and that rectal cancers with high α-SMA/epithelial area ratio had low proliferation rates. Interestingly, the α-SMA/epithelial area ratio was an adverse prognostic factor with regard to recurrence-free survival in univariate analysis. In addition, multivariate analysis showed that an α-SMA/epithelial area ratio above 1 provides an independent prognostic value associated with a poor recurrence-free survival. CONCLUSION: These results suggest that neoadjuvant treatment has an impact on CAFs in rectal cancer. The correlation of CAFs with decreased recurrence-free survival and abundant experimental data in the literature suggest that under certain circumstances, not yet very well understood, CAFs may favour tumour progression.

X-ray radiation promotes the metastatic potential of tongue squamous cell carcinoma cells via modulation of biomechanical and cytoskeletal properties.

This study investigated the metastatic potential of tongue squamous cell carcinoma (TSCC) cells after X-ray irradiation as well as radiation-induced changes in the biomechanical properties and cytoskeletal structure that are relevant to metastasis. Tca-8113 TSCC cells were X-ray-irradiated at increasing doses (0, 1, 2, or 4 Gy), and 24 h later, migration was evaluated with the wound healing and transwell migration assays, while invasion was assessed with the Matrigel invasion assay. Confocal and atomic force microscopy were used to examine changes in the structure of the actin cytoskeleton and Young's modulus (cell stiffness), respectively. X-ray radiation induced dose-dependent increases in invasive and migratory potentials of cells relative to unirradiated control cells (p < 0.05). The Young's modulus of irradiated cells was decreased by radiation exposure (p < 0.05), which was accompanied by alterations in the integrity and organization of the cytoskeletal network, as evidenced by a decrease in the signal intensity of actin fibers (p < 0.05). X-ray irradiation enhanced migration and invasiveness in Tca-8113 TSCC cells by altering their biomechanical properties and the organization of the actin cytoskeleton. A biomechanics-based analysis can provide an additional platform for assessing tumor response to radiation and optimization of cancer therapies.

The number of accumulated photons and the quality of stimulated emission depletion lifetime images.

Time binning is used to increase the number of photon counts in the peak channel of stimulated emission depletion fluorescence lifetime decay curves to determine how it affects the resulting lifetime image. The fluorescence lifetime of the fluorophore, Alexa Fluor 594 phalloidin, bound to F-actin is probed in cultured S2 cells at a spatial resolution of ~40 nm. This corresponds to a 10-fold smaller probe volume compared to confocal imaging, and a reduced number of photons contributing to the signal. Pixel-by-pixel fluorescence lifetime measurements and error analysis show that an average of 40 ± 30 photon counts in the peak channel with a signal-to-noise ratio of 20 is enough to calculate a reliable fluorescence lifetime from a single exponential fluorescence decay. No heterogeneity in the actin cytoskeleton in different regions of the cultured cells was measured in the 40-400 nm spatial regime.

Intermittent depolymerization of actin filaments is caused by photo-induced dimerization of actin protomers.

Actin, one of the most abundant proteins within eukaryotic cells, assembles into long filaments that form intricate cytoskeletal networks and are continuously remodelled via cycles of actin polymerization and depolymerization. These cycles are driven by ATP hydrolysis, a process that also acts to destabilize the filaments as they grow older. Recently, abrupt dynamical changes during the depolymerization of single filaments have been observed and seemed to imply that old filaments are more stable than young ones [Kueh HY, et al. (2008) Proc Natl Acad Sci USA 105:16531-16536]. Using improved experimental setups and quantitative theoretical analysis, we show that these abrupt changes represent actual pauses in depolymerization, unexpectedly caused by the photo-induced formation of actin dimers within the filaments. The stochastic dimerization process is triggered by random transitions of single, fluorescently labeled protomers. Each pause represents the delayed dissociation of a single actin dimer, and the statistics of these single molecule events can be determined by optical microscopy. Unlabeled actin filaments do not exhibit pauses in depolymerization, which implies that, in vivo, older filaments become destabilized by ATP hydrolysis, unless this aging effect is overcompensated by actin-binding proteins. The latter antagonism can now be systematically studied for single filaments using our combined experimental and theoretical method. Furthermore, the dimerization process discovered here provides a molecular switch, by which one can control the length of actin filaments via changes in illumination. This process could also be used to locally "freeze" the dynamics within networks of filaments.

Cardiac endothelial cells isolated from mouse heart - a novel model for radiobiology.

Cardiovascular disease is recognized as an important clinical problem in radiotherapy and radiation protection. However, only few radiobiological models relevant for assessment of cardiotoxic effects of ionizing radiation are available. Here we describe the isolation of mouse primary cardiac endothelial cells, a possible target for cardiotoxic effects of radiation. Cells isolated from hearts of juvenile mice were cultured and irradiated in vitro. In addition, cells isolated from hearts of locally irradiated adult animals (up to 6 days after irradiation) were tested. A dose-dependent formation of histone γH2A.X foci was observed after in vitro irradiation of cultured cells. However, such cells were resistant to radiation-induced apoptosis. Increased levels of actin stress fibres were observed in the cytoplasm of cardiac endothelial cells irradiated in vitro or isolated from irradiated animals. A high dose of 16 Gy did not increase permeability to Dextran in monolayers formed by endothelial cells. Up-regulated expression of Vcam1, Sele and Hsp70i genes was detected after irradiation in vitro and in cells isolated few days after irradiation in vivo. The increased level of actin stress fibres and enhanced expression of stress-response genes in irradiated endothelial cells are potentially involved in cardiotoxic effects of ionizing radiation.

Influence of pulsed magnetic fields on the morphology of bone cells in early stages of growth.

The effect of electromagnetic fields on living systems has been studied both in vivo and in vitro in a wide range of organisms, cells and tissues. However, the mechanism of action of electromagnetic fields is not yet clearly defined. This paper presents the results of applying a pulsed magnetic field of 70ms width, intensity of 0.65mT at 4Hz in human osteoblasts, during 45min. The magnetic field application was conducted on crops of both 24 and 48h of proliferation. The effect of applying magnetic fields was assessed using parameters such as cell density, protein content, distribution of F-actin fibrils and ß-tubulin and integrity of nuclear structure. The results indicate no alteration in either protein synthesis or nuclear structure, or in the number of cells. However, we observed that exposure to these fields induces changes in the distribution of cytoskeletal proteins of osteoblasts.

Keratinocyte growth factor down-regulates intracellular ROS production induced by UVB.

BACKGROUND: Exposure to ultraviolet (UV) radiation causes a complex cellular response, mostly mediated by the production of reactive oxygen species (ROS), which can be counteracted by exogenous treatments and endogenous mechanisms with anti-oxidant and scavenger properties. Keratinocyte growth factor (KGF/FGF7), a member of the fibroblast growth factor family, promotes epithelial growth and differentiation and is involved in cell survival after oxidant injuries. OBJECTIVE: We analyzed the role of KGF in the control of intracellular ROS production and oxidative stress after UVB exposure on KGF receptor (KGFR) transfected cells and human immortalized and primary keratinocytes. METHODS: We assessed the intracellular ROS production measuring the intensity of the oxidation-sensitive fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA) by confocal microscopy, as well as the catalase activity by spectrophotometric assay. Moreover, morphological and biochemical analysis of actin cytoskeleton reorganization was evaluated as a further marker of oxidative damage. RESULTS: Our data show that KGF significantly reduces intracellular ROS generation in response to UVB, preserves the decrease of catalase activity and prevents actin cytoskeleton rearrangement. CONCLUSION: Our results provide a further evidence that KGF may be crucial for an efficient skin photoprotection, demonstrating a direct role for KGF in the reduction of intracellular ROS content following UVB exposure.

Photoinduced in situ formation of various F-actin assemblies with a photoresponsive polycation.

We developed a novel in situ method for the control of F-actin assembly by using a synthetic photoresponsive polycation. The photoresponsive polycation mainly comprises a water-soluble cationic monomer and also contains a small amount of the monomer of a triphenylmethane leucohydroxide derivative (20 mol %), which is a well-known photochromic molecule that can be cationized in aqueous solution by ultra violet (UV) irradiation, thereby causing an increase in the total charge on the photoresponsive polycation. Thus, by exposure to UV radiation in aqueous solution, F-actin and the photoresponsive polycation start assembling into F-actin/photoresponsive polycation complexes of various morphologies such as bundles, coils, and networks, depending upon the concentrations of both the F-actin and salt. Further, localized UV irradiation can be applied in order to control the local formation of F-actin/photoresponsive polycation complexes. Thus, this technique provides a novel method for the spatiotemporal control of F-actin assembly and can be applied to investigate the unknown characteristics of F-actin.

High-grade glioma before and after treatment with radiation and Avastin: initial observations.

We evaluate the effects of adjuvant treatment with the angiogenesis inhibitor Avastin (bevacizumab) on pathological tissue specimens of high-grade glioma. Tissue from five patients before and after treatment with Avastin was subjected to histological evaluation and compared to four control cases of glioma before and after similar treatment protocols not including bevacizumab. Clinical and radiographic data were reviewed. Histological analysis focused on microvessel density and vascular morphology, and expression patterns of vascular endothelial growth factor-A (VEGF-A) and the hematopoietic stem cell, mesenchymal, and cell motility markers CD34, smooth muscle actin, D2-40, and fascin. All patients with a decrease in microvessel density had a radiographic response, whereas no response was seen in the patients with increased microvessel density. Vascular morphology showed apparent "normalization" after Avastin treatment in two cases, with thin-walled and evenly distributed vessels. VEGF-A expression in tumor cells was increased in two cases and decreased in three and did not correlate with treatment response. There was a trend toward a relative increase of CD34, smooth muscle actin, D2-40, and fascin immunostaining following treatment with Avastin. Specimens from four patients with recurrent malignant gliomas before and after adjuvant treatment (not including bevacizumab) had features dissimilar from our study cases. We conclude that a change in vascular morphology can be observed following antiangiogenic treatment. There seems to be no correlation between VEGF-A expression and clinical parameters. While the phenomena we describe may not be specific to Avastin, they demonstrate the potential of tissue-based analysis for the discovery of clinically relevant treatment response biomarkers.

Reduction of photobleaching and photodamage in single molecule detection: observing single actin monomer in skeletal myofibrils.

Recent advances in detector technology make it possible to achieve single molecule detection (SMD) in a cell. SMD avoids complications associated with averaging signals from large assemblies and with diluting and disorganizing proteins. However, it requires that cells be illuminated with an intense laser beam, which causes photobleaching and cell damage. To reduce these effects, we study cells on coverslips coated with silver nanoparticle monolayers (NML). Muscle is used as an example. Actin is labeled with a low concentration of fluorescent phalloidin to assure that less than a single molecule in a sarcomere is fluorescent. On a glass substrate, the fluorescence of actin decays in a step-wise fashion, establishing a single molecule detection regime. Single molecules of actin in living muscle are visualized for the first time. NML coating decreases the fluorescence lifetime 17 times and enhances intensity ten times. As a result, fluorescence of muscle bleaches four to five times slower than on glass. Monolayers decrease photobleaching because they shorten the fluorescence lifetime, thus decreasing the time that a fluorophore spends in the excited state when it is vulnerable to oxygen attack. They decrease damage to cells because they enhance the electric field near the fluorophore, making it possible to illuminate samples with weaker light.

Electronically activated actin protein polymerization and alignment.

Biological systems are the paragon of dynamic self-assembly, using a combination of spatially localized protein complexation, ion concentration, and protein modification to coordinate a diverse set of self-assembling components. Biomimetic materials based upon biologically inspired design principles or biological components have had some success at replicating these traits, but have difficulty capturing the dynamic aspects and diversity of biological self-assembly. Here, we demonstrate that the polymerization of ion-sensitive proteins can be dynamically regulated using electronically enhanced ion mixing and monomer concentration. Initially, the global activity of the cytoskeletal protein actin is inhibited using a low-ionic strength buffer that minimizes ion complexation and protein-protein interactions. Nucleation and growth of actin filaments are then triggered by a low-frequency AC voltage, which causes local enhancement of the actin monomer concentration and mixing with Mg(2+). The location and extent of polymerization are governed by the voltage and frequency, producing highly ordered structures unprecedented in bulk experiments. Polymerization rate and filament orientation could be independently controlled using a combination of low-frequency (approximately 100 Hz) and high frequency (1 MHz) AC voltages, creating a range of macromolecular architectures from network hydrogel microparticles to highly aligned arrays of actin filaments with approximately 750 nm periodicity. Since a wide range of proteins are activated upon complexation with charged species, this approach may be generally applicable to a variety of biopolymers and proteins.

Radiation effects on the cytoskeleton of endothelial cells and endothelial monolayer permeability.

PURPOSE: To investigate the effects of radiation on the endothelial cytoskeleton and endothelial monolayer permeability and to evaluate associated signaling pathways, which could reveal potential mechanisms of known vascular effects of radiation. METHODS AND MATERIALS: Cultured endothelial cells were X-ray irradiated, and actin filaments, microtubules, intermediate filaments, and vascular endothelial (VE)-cadherin junctions were examined by immunofluorescence. Permeability was determined by the passage of fluorescent dextran through cell monolayers. Signal transduction pathways were analyzed using RhoA, Rho kinase, and stress-activated protein kinase-p38 (SAPK2/p38) inhibitors by guanosine triphosphate-RhoA activation assay and transfection with RhoAT19N. The levels of junction protein expression and phosphorylation of myosin light chain and SAPK2/p38 were assessed by Western blotting. The radiation effects on cell death were verified by clonogenic assays. RESULTS: Radiation induced rapid and persistent actin stress fiber formation and redistribution of VE-cadherin junctions in microvascular, but not umbilical vein endothelial cells, and microtubules and intermediate filaments remained unaffected. Radiation also caused a rapid and persistent increase in microvascular permeability. RhoA-guanosine triphosphatase and Rho kinase were activated by radiation and caused phosphorylation of downstream myosin light chain and the observed cytoskeletal and permeability changes. SAPK2/p38 was activated by radiation but did not influence either the cytoskeleton or permeability. CONCLUSION: This study is the first to show rapid activation of the RhoA/Rho kinase by radiation in endothelial cells and has demonstrated a link between this pathway and cytoskeletal remodeling and permeability. The results also suggest that the RhoA pathway might be a useful target for modulating the permeability and other effects of radiation for therapeutic gain.

Nanosecond pulsed electric fields have differential effects on cells in the S-phase.

Nanosecond pulsed electric fields (nsPEFs) are a type of nonthermal, nonionizing radiation that exhibit intense electric fields with high power, but low energy. NsPEFs extend conventional electroporation (EP) to affect intracellular structures and functions and depending on the intensity, can induce lethal and nonlethal cell signaling. In this study, HCT116 human colon carcinoma cells were synchronized to the S-phase or remained unsynchronized, exposed to electric fields of 60 kV/cm with either 60-ns or 300-ns durations, and analyzed for apoptosis and proliferative markers. Several nsPEF structural and functional targets were identified. Unlike unsynchronized cells, S-phase cells under limiting conditions exhibited greater membrane integrity and caspase activation and maintained cytoskeletal structure. Regardless of synchronization, cells exposed to nsPEFs under these conditions primarily survived, but exhibited some turnover and delayed proliferation in cell populations, as well as reversible increases in phosphatidylserine externalization, membrane integrity, and nuclei size. These results show that nsPEFs can act as a nonligand agonist to modulate plasma membrane (PM) and intracellular structures and functions, as well as differentially affect cells in the S-phase, but without effect on cell survival. Furthermore, nsPEF effects on the nucleus and cytoskeleton may provide synergistic therapeutic actions with other agents, such as ionizing radiation or chemotherapeutics that affect these same structures.

Ultraviolet radiation (UV) induces reorganization of actin cytoskeleton in CHOAA8 cells.

Effect of UV radiation on actin cytoskeleton was studied in CHOAA8 cells by fluorescence and electron microscopy. UV irradiated cells showed impaired adherence, disruption of the actin filaments and stronger F-actin labeling in the center of the cell. Attached cells, especially enlarged ones showed rather weak labeling of stress fibers and bundles of F-actin in the cytoplasm, but some cells with intensive labeling of these structures were also observed. Detached cells were rounded, showed strong F-actin labeling and often had buds. At the ultrastructural level UV-irradiated cells showed segmented nuclei, bodies resembling micronuclei, dilatation of endoplasmic reticulum, swollen and disturb mitochondria. Immunogold labeling of actin at the ultrastructural level was observed in non-radiated and UV irradiated cells. Actin labeling was seen in nuclei and cytoplasm. In nuclei gold particles were localized in the area of condense chromatin. Labeling for actin was not found after control incubation. Our observations show that UV radiation promotes changes in the distribution of actin in CHOAA8 cells. The results also suggest that not only reorganization of actin but changes in organelles are involved in the process of apoptosis initiated by UV radiation.