Selenium nanoparticles induced membrane bio-mechanical property changes in MCF-7 cells by disturbing membrane molecules and F-actin

Selenium nanoparticles (Se NPs) have been served as promising materials for biomedical applications, especially for cancer treatment. The anti-cancer effects of Se NPs against cancer cells have been widely studied in recent years, but whether Se NPs can induce the changes of cell membrane bio-mechanical properties in cancer cells still remain unexplored. In this Letter, we prepared Se NPs for investigating the intracellular localization of Se NPs in MCF-7 cells and determined the effects of Se NPs on apoptosis and necrosis in MCF-7 cells. Especially, we reported for the first time about the effects of Se NPs on the bio-mechanical properties of cancer cells and found that Se NPs could remarkably decrease the adhesion force and Young’s modulus of MCF-7 cells. To further understand the potential mechanisms about how Se NPs affect the bio-mechanical properties of MCF-7 cells, we also investigated the expression of CD44 molecules, the structure and the amounts of F-actin. The results indicated that the decreased adhesion force between AFM tip and cell membrane was partially due to the changes of membrane molecules induced by Se NPs, such as the down-regulation of trans-membrane CD44 molecules. Additionally, the decrease of Young’s modulus of MCF-7 cells was due to the dis-organization and down-regulation of F-actin induced by Se NPs. These results collectively suggested that cell membrane was of vital importance in Se NPs induced toxicity in cancer cells, which could be served as a potential target for cancer treatment by Se NPs.     

Sildenafil protects epithelial cell through the inhibition of xanthine oxidase and the impairment of ROS production

Abstract

Xanthine oxidase (XO) plays an important role in various forms of ischemic and vascular injuries, inflammatory diseases and chronic heart failure. The XO inhibitors allopurinol and oxypurinol held considerable promise in the treatment of these conditions both in experimental animals and in human clinical trials. More recently, an endothelium-based protective effect of sildenafil has been reported in preconditioning prior to ischemia/reperfusion in healthy human subjects. Based on the structural similarities between allopurinol and oxypurinol with sildenafil and with zaprinast the authors have investigated the potential effects of these latter compounds on the buttermilk XO and on non-tumourigenic (HMEC) and malignant (MCF7) human mammary epithelial cells. Both sildenafil and zaprinast induced a significant and consistent decrease of XO expression and activity in either cell line. In MCF7 cells only, this effect was associated with the abrogation of xanthine-induced cytotoxicity. Overall, the data suggest that the protective effect of sildenafil on epithelial cells is a consequence of the inhibition of the XO and of the resulting decrease of free oxygen radical production that may influence the expression of NADPH oxidase and PDE-5.     

原子力显微镜在细胞弹性研究中的应用

细胞表面的力学性质会随着细胞所处环境的不同而发生改变,它的变化间接反映出胞内复杂的生理过程。原子力显微镜（atomic force microscope,AFM）能以高的灵敏度和分辨率检测活体细胞,通过利用赫兹模型分析力曲线可以获得细胞的弹性信息。本文简介了原子力显微镜的工作原理与工作模式,着重介绍利用AFM力曲线检测细胞弹性的方法及其在细胞运动、细胞骨架、细胞黏附、细胞病理等方面的应用成果,表明AFM已经成为细胞弹性研究中十分重要的显微技术。     

Quantitative cellular uptake, localization and cytotoxicity of curcumin in normal and tumor cells

Using absorption and fluorescence spectroscopic methods, quantitative cellular uptake of curcumin, an antioxidant and anti-tumor agent from Curcuma longa, was calculated in two types of normal cells: spleen lymphocytes, and NIH3T3 and two tumor cell lines: EL4 and MCF7. Both the uptake and fluorescence intensity of curcumin were significantly higher in tumor cells compared to the normal cells. A linear dependency on the uptake was observed with treatment concentration of curcumin. Using laser confocal microscopy, intracellular localization of curcumin was monitored and the results indicated that curcumin is located both in the cell membrane and the nucleus. Sub-cellular fractionation of curcumin-loaded MCF7 cells supported the differential distribution of curcumin in membrane, cytoplasm and nuclear compartments of cell with maximum localization in the membrane. Cytotoxicity studies in different cell lines indicated that the toxicity of curcumin increased with increasing uptake.     

Age-Related Changes in the Mechanical Properties of Human Fibroblasts and Its Prospective Reversal After Anti-Wrinkle Tripeptide Treatment

One of an essential characteristic of human skin are time dependent mechanical properties. Here, we demonstrate that stiffness of human dermal fibroblast correlates with age and it can be restored after anti-wrinkle tripeptide treatment. The stiffness of human fibroblasts isolated from donors of 30-, 40- and 60 years old were examined. Additionally the effect of anti- wrinkle tripeptide of latter cells was investigated. The atomic force microscopy measurements were performed on untreated fibroblast as well as on treated with the peptide. The Young’s modulus for two indentation depths 200 and 600 nm of each cell type was determined. The Young’s modulus increases with age of the cells. The highest values of Young’s modulus were obtained for fibroblasts collected from 60 years old donors, for indentation depth of ~200 nm. For larger indentation depth of 600 nm there are no significant differences in stiffness between cells. Fibroblasts treated with the anti-wrinkle tripeptide exhibit lower Young’s modulus. The cells derived from 40- and 60-years old donors restored stiffness characteristic to the level of 30 years old subjects. The results show correlation between stiffness and age of the human fibroblast as well as impact of anti-wrinkle tripeptide on the mechanical properties of skin cells.     

The Influence of Physical and Physiological Cues on Atomic Force Microscopy-Based Cell Stiffness Assessment

Atomic force microscopy provides a novel technique for differentiating the mechanical properties of various cell types. Cell elasticity is abundantly used to represent the structural strength of cells in different conditions. In this study, we are interested in whether physical or physiological cues affect cell elasticity in Atomic force microscopy (AFM)-based assessments. The physical cues include the geometry of the AFM tips, the indenting force and the operating temperature of the AFM. All of these cues show a significant influence on the cell elasticity assessment. Sharp AFM tips create a two-fold increase in the value of the effective Young’s modulus (E_eff) relative to that of the blunt tips. Higher indenting force at the same loading rate generates higher estimated cell elasticity. Increasing the operation temperature of the AFM leads to decreases in the cell stiffness because the structure of actin filaments becomes disorganized. The physiological cues include the presence of fetal bovine serum or extracellular matrix-coated surfaces, the culture passage number, and the culture density. Both fetal bovine serum and the extracellular matrix are critical for cells to maintain the integrity of actin filaments and consequently exhibit higher elasticity. Unlike primary cells, mouse kidney progenitor cells can be passaged and maintain their morphology and elasticity for a very long period without a senescence phenotype. Finally, cell elasticity increases with increasing culture density only in MDCK epithelial cells. In summary, for researchers who use AFM to assess cell elasticity, our results provide basic and significant information about the suitable selection of physical and physiological cues.     

Temporal analysis of vascular smooth muscle cell elasticity and adhesion reveals oscillation waveforms that differ with aging

A spectral analysis approach was developed for detailed study of time‐resolved, dynamic changes in vascular smooth muscle cell (VSMC) elasticity and adhesion to identify differences in VSMC from young and aged monkeys. Atomic force microscopy (AFM) was used to measure Young’s modulus of elasticity and adhesion as assessed by fibronectin (FN) or anti‐beta 1 integrin interaction with the VSMC surface. Measurements demonstrated that VSMC cells from old vs. young monkeys had increased elasticity (21.6 kPa vs. 3.5 kPa or a 612% increase in elastic modulus) and adhesion (86 pN vs. 43 pN or a 200% increase in unbinding force). Spectral analysis identified three major frequency components in the temporal oscillation patterns for elasticity (ranging from 1.7 × 10^?3 to 1.9 × 10^?2 Hz in old and 8.4 × 10^?4 to 1.5 × 10^?2 Hz in young) and showed that the amplitude of oscillation was larger (P < 0.05) in old than in young at all frequencies. It was also observed that patterns of oscillation in the adhesion data were similar to the elasticity waveforms. Cell stiffness was reduced and the oscillations were inhibited by treatment with cytochalasin D, ML7 or blebbistatin indicating the involvement of actin–myosin‐driven processes. In conclusion, these data demonstrate the efficacy of time‐resolved analysis of AFM cell elasticity and adhesion measurements and that it provides a uniquely sensitive method to detect real‐time functional differences in biomechanical and adhesive properties of cells. The oscillatory behavior suggests that mechanisms governing elasticity and adhesion are coupled and affected differentially during aging, which may link these events to changes in vascular stiffness.     

Dynamic effect of beta-amyloid 42 on cell mechanics

Aβ_1-42, which is highly toxic to neural cells, is commonly present in the brains of people with Alzheimer’s disease. In this study, dynamic changes in cell mechanics were monitored under Aβ-induced toxicity. To investigate the changes in cellular mechanical properties, we used Aβ_1-42 oligomer at different concentrations to treat human neuroblastoma SH-SY5H cells. Results demonstrated a two-stage dynamic change in cell mechanics during neurodegeneration. Additionally, Young’s modulus (YM) of the treated cells increased in a short period. The reasons include alteration in surface tension, osmotic pressure, and actin polymerization. Rough cellular membranes were observed from atomic force microscope (AFM) measurement. However, the cellular YM gradually decreased when the cells were continuously exposed to Aβ_1-42 or to a high concentration of Aβ_1-42. The major reason for the decreased YM was microtubule disassembly. Dynamic change in YM reflects different activities in cytoplasm in response to Aβ_1-42. The characteristic changes in cell mechanics provided insights into the dynamic neurodegeneration process of cells induced by Aβ_1-42 oligomer.     

Characterization of Dynamic Behaviour of MCF7 and MCF10A Cells in Ultrasonic Field Using Modal and Harmonic Analyses

Treatment options specifically targeting tumour cells are urgently needed in order to reduce the side effects accompanied by chemo- or radiotherapy. Differences in subcellular structure between tumour and normal cells determine their specific elasticity. These structural differences can be utilised by low-frequency ultrasound in order to specifically induce cytotoxicity of tumour cells. For further evaluation, we combined in silico FEM (finite element method) analyses and in vitro assays to bolster the significance of low-frequency ultrasound for tumour treatment. FEM simulations were able to calculate the first resonance frequency of MCF7 breast tumour cells at 21 kHz in contrast to 34 kHz for the MCF10A normal breast cells, which was due to the higher elasticity and larger size of MCF7 cells. For experimental validation of the in silico-determined resonance frequencies, equipment for ultrasonic irradiation with distinct frequencies was constructed. Differences for both cell lines in their response to low-frequent ultrasonic treatment were corroborated in 2D and in 3D cell culture assays. Treatment with ~ 24.5 kHz induced the death of MCF7 cells and MDA-MB-231 metastases cells possessing a similar elasticity; frequencies of > 29 kHz resulted in cytotoxicity of MCF10A. Fractionated treatments by ultrasonic irradiation of suspension myeloid HL60 cells resulted in a significant decrease of viable cells, mostly significant after threefold irradiation in intervals of 3 h. Most importantly in regard to a clinical application, combined ultrasonic treatment and chemotherapy with paclitaxel showed a significantly increased killing of MCF7 cells compared to both monotherapies. In summary, we were able to determine for the first time for different tumour cell lines a specific frequency of low-intensity ultrasound for induction of cell ablation. The cytotoxic effect of ultrasonic irradiation could be increased by either fractionated treatment or in combination with chemotherapy. Thus, our results will open new perspectives in tumour treatment.     

Heat shock protein 70 and glycoprotein 96 are differentially expressed on the surface of malignant and nonmalignant breast cells

Heat shock proteins (HSPs), which are important for a number of different intracellular functions, are occasionally found on the surface of cells. The function of heat shock protein on the cell surface is not understood, although it has been shown to be greater in some tumor cells and some virally infected cells. Surface expression of both glycoprotein 96 (gp96) and Hsp70 occurs on tumor cells, and this expression correlates with natural killer cell killing of the cells. We examined the surface expression of gp96 and Hsp70 on human breast cell lines MCF7, MCF10A, AU565, and HS578, and in primary human mammary epithelial cells by immunofluorescence microscopy and flow cytometry. The nonmalignant cell lines HS578, MCF10A, and HMEC showed no surface expression of gp96, whereas malignant cell lines MCF7 and AU565 were positive for gp96 surface expression. All of the breast cell lines examined showed Hsp70 surface expression. These results also confirm previous studies, demonstrating that Hsp70 is on the plasma membrane of tumor cell lines. Given the involvement of heat shock proteins, gp96 and Hsp70, in innate and adaptive immunity, these observations may be important in the immune response to tumor cells.     

Dietary antioxidant curcumin inhibits microtubule assembly through tubulin binding

Curcumin, a component of turmeric, has potent antitumor activity against several tumor types. However, its molecular target and mechanism of antiproliferative activity are not clear. Here, we identified curcumin as a novel antimicrotubule agent. We have examined the effects of curcumin on cellular microtubules and on reconstituted microtubules in vitro. Curcumin inhibited HeLa and MCF-7 cell proliferation in a concentration-dependent manner with IC(50) of 13.8 +/- 0.7 microm and 12 +/- 0.6 microm, respectively. At higher inhibitory concentrations (> 10 microm), curcumin induced significant depolymerization of interphase microtubules and mitotic spindle microtubules of HeLa and MCF-7 cells. However, at low inhibitory concentrations there were minimal effects on cellular microtubules. It disrupted microtubule assembly in vitro, reduced GTPase activity, and induced tubulin aggregation. Curcumin bound to tubulin at a single site with a dissociation constant of 2.4 +/- 0.4 microm and the binding of curcumin to tubulin induced conformational changes in tubulin. Colchicine and podophyllotoxin partly inhibited the binding of curcumin to tubulin, while vinblastine had no effect on the curcumin-tubulin interactions. The data together suggested that curcumin may inhibit cancer cells proliferation by perturbing microtubule assembly dynamics and may be used to develop efficacious curcumin analogues for cancer chemotherapy.     

The geodiamolide H, derived from Brazilian sponge Geodia corticostylifera, regulates actin cytoskeleton, migration and invasion of breast cancer cells cultured in three-dimensional environment

We are investigating effects of the depsipeptide geodiamolide H, isolated from the Brazilian sponge Geodia corticostylifera, on cancer cell lines grown in 3D environment. As shown previously geodiamolide H disrupts actin cytoskeleton in both sea urchin eggs and breast cancer cell monolayers. We used a normal mammary epithelial cell line MCF 10A that in 3D assay results formation of polarized spheroids. We also used cell lines derived from breast tumors with different degrees of differentiation: MCF7 positive for estrogen receptor and the Hs578T, negative for hormone receptors. Cells were placed on top of Matrigel. Spheroids obtained from these cultures were treated with geodiamolide H. Control and treated samples were analyzed by light and confocal microscopy. Geodiamolide H dramatically affected the poorly differentiated and aggressive Hs578T cell line. The peptide reverted Hs578T malignant phenotype to polarized spheroid-like structures. MCF7 cells treated by geodiamolide H exhibited polarization compared to controls. Geodiamolide H induced striking phenotypic modifications in Hs578T cell line and disruption of actin cytoskeleton. We investigated effects of geodiamolide H on migration and invasion of Hs578T cells. Time-lapse microscopy showed that the peptide inhibited migration of these cells in a dose-dependent manner. Furthermore invasion assays revealed that geodiamolide H induced a 30% decrease on invasive behavior of Hs578T cells. Our results suggest that geodiamolide H inhibits migration and invasion of Hs578T cells probably through modifications in actin cytoskeleton. The fact that normal cell lines were not affected by treatment with geodiamolide H stimulates new studies towards therapeutic use for this peptide.     

Escherichia coli lipopolysaccharide induces alveolar epithelial cell stiffening

IntroductionApplication of lipopolysaccharide (LPS) is a widely employed model to mimic acute respiratory distress syndrome (ARDS). Available data regarding LPS-induced biomechanical changes on pulmonary epithelial cells are limited only to P. aeruginosa LPS. Considering that LPS from different bacteria could promote a specific mechanical response in epithelial cells, we aim to assess the effect of E. coli LPS, widely employed as a model of ARDS, in the biomechanics of alveolar epithelial cells.MethodsYoung’s modulus (E) of alveolar epithelial cells (A549) was measured by atomic force microscopy every 5 min throughout 60 min of experiment after treatment with LPS from E. coli (100 μg/mL). The percentage of cells presenting actin stress fibers (F-actin staining) was also evaluated. Control cells were treated with culture medium and the values obtained were compared with LPS-treated cells for each time-point.ResultsApplication of LPS induced significant increase in E after 20 min (77%) till 60 min (104%) in comparison to controls. Increase in lung epithelial cell stiffness induced by LPS was associated with a higher number of cells presenting cytoskeletal remodeling.ConclusionsThe observed effects of E. coli LPS on alveolar epithelial cells suggest that this widely-used LPS is able to promote a quick formation of actin stress fibers and stiffening cells, thereby facilitating the disruption of the pulmonary epithelial barrier.     

Distinct signaling pathways regulate differential opioid effects on actin cytoskeleton in malignant MCF7 and nonmalignant MCF12A human breast epithelial cells

The mechanisms through which opioids regulate the activity of malignant breast epithelial cells are currently unknown. In the present study we report the differential actin cytoskeleton reorganization induced by opioids in malignant (MCF7) and nonmalignant (MCF12A) breast epithelial cells expressing functional opioid receptors. Exposure of MCF7 cells to the opioid agonist alpha(s1) casomorphin induced important actin assembly and reorganization, including the formation of filopodia and lamellipodia. In contrast, incubation of MCF12A cells with alpha(s1) casomorphin revealed a partial but transient disassembly of actin microfilaments. Immunoprecipitation and immunoblot analyses showed rapid phosphorylation of focal adhesion kinase (FAK) and vinculin in opioid-treated MCF7 cells. Moreover, FAK associates with phosphatidylinositol-3 (PI-3 kinase), the latter being subsequently phosphorylated and activated. In addition, a substantial activation of the small GTPase Rac1 was observed. Pretreatment of MCF7 cells with the specific PI-3 kinase inhibitor wortmannin abolished both the activation of Rac1 and actin reorganization, while the opioid-induced phosphorylation of FAK and vinculin remained unaffected. Interestingly, in opioid-treated MCF12A cells this signaling cascade remained inactive, while we identified rapid phosphorylation of actin regulating the protein villin. Finally, opioids differentially inhibited cell motility in each cell line. Our data suggest a distinct, opioid-induced, signaling pathway activated in malignant breast epithelial cells, leading to important actin reorganization. These findings may indicate a potential antineoplastic role of opiates, based on the activation of differential signaling mechanisms.     

The regulatory effect of Tau protein on polymerization of MCF7 microtubules in vitro

Growing evidence continues to point toward the critical role of beta tubulin isotypes in regulating some intracellular functions. Changes that were observed in the microtubules’ intrinsic dynamics, the way they interact with some chemotherapeutic agents, or differences on translocation specifications of some molecular motors along microtubules, were associated to their structural uniqueness in terms of beta tubulin isotype distributions. These findings suggest that the effects of microtubule associated proteins (MAPs) may also vary on structurally different microtubules. Among different microtubule associated proteins, Tau proteins, which are known as neuronal MAPs, bind to beta tubulin, stabilize microtubules, and consequently promote their polymerizations.In this study, in a set of well controlled experiments, the direct effect of Tau proteins on the polymerization of two structurally different microtubules, porcine brain and breast cancer (MCF7), were tested and compared. Remarkably, we found that in contrast with the promoted effect of Tau proteins on brain microtubules’ polymerization, MCF7 expressed a demoted polymerization while interacting with Tau proteins. This finding can potentially be a novel insight into the mechanism of drug resistance in some breast cancer cells.It has been reported that microtubules show destabilizing behavior in some MCF7 cells with overexpression of Tau protein when treated with a microtubules’ stabilizing agent, Taxol. This behavior has been classified by others as drug resistance, but it may instead be potentially caused by a competition between the destabilizing effect of the Tau protein and the stabilizing effect of the drug on MCF7 microtubules. Also, we quantified the polarization coefficient of MCF7 microtubules in the presence and absence of Tau proteins by the electro-orientation method and compared the values. The two significantly different values obtained can possibly be one factor considered to explain the effect of Tau proteins on the polymerization of MCF7 microtubules.     

Biophysical Characterization of Bladder Cancer Cells with Different Metastatic Potential

Specific membrane capacitance (SMC) and Young’s modulus are two important parameters characterizing the biophysical properties of a cell. In this work, the SMC and Young’s modulus of two cell lines, RT4 and T24, corresponding to well differentiated (low grade) and poorly differentiated (high grade) urothelial cell carcinoma (UCC), respectively, were quantified using microfluidic and AFM measurements. Quantitative differences in SMC and Young’s modulus values of the high-grade and low-grade UCC cells are, for the first time, reported.     

Effect of curcumin extract against oxidative stress on both structure and deformation capability of red blood cell

The normal deformability of erythrocytes plays an important role in ensuring blood mobility, erythrocyte longevity, and microcirculation, which is the ability of erythrocytes to change shapes in response to external forces. However, the effects of curcumin extracts on the deformability of erythrocytes have not yet been evaluated. Accordingly, in this study, we explored the effects of pre-treatment with curcumin extract on erythrocyte deformation and erythrocyte band 3 (SLC4A1; EB3) expression. We also evaluated the associations between EB3 expression and erythrocyte deformability induced by hydrogen peroxide. Blood samples were divided into the control group, pre-treatment group (treated with curcumin extract or vitamin C), and negative control group, and oxidant stress parameters, antioxidant status, erythrocyte deformability and elasticity, and EB3 modifications were evaluated using immunoblotting and immunofluorescence staining. Hydrogen peroxide significantly increased oxidative stress parameters, modulus elasticity values and clustered EB3 levels and induced conjugation of membrane proteins to form high-molecular-weight complexes (p < 0.05). Erythrocyte deformability and elasticity were significantly decreased in the treated groups compared with those in the control group. Overall, our findings suggested that pre-treatment with curcumin extracts increased antioxidant status, reduced EB3 cross-linking, and improved erythrocyte deformability, to an even better extent than vitamin C. These results provide important insights into the effects of treatment with curcumin extracts on erythrocyte damage and suggest that curcumin may have applications in antioxidant therapy.