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1.
J Cell Sci ; 135(5)2022 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33414166

RESUMO

Ferroptosis is a regulated, non-apoptotic form of cell death, characterized by hydroxy-peroxidation of discrete phospholipid hydroperoxides, particularly hydroperoxyl (Hp) forms of arachidonoyl- and adrenoyl-phosphatidylethanolamine, with a downstream cascade of oxidative damage to membrane lipids, proteins and DNA, culminating in cell death. We recently showed that human trophoblasts are particularly sensitive to ferroptosis caused by depletion or inhibition of glutathione peroxidase 4 (GPX4) or the lipase PLA2G6. Here, we show that trophoblastic ferroptosis is accompanied by a dramatic change in the trophoblast plasma membrane, with macro-blebbing and vesiculation. Immunofluorescence revealed that ferroptotic cell-derived blebs stained positive for F-actin, but negative for cytoplasmic organelle markers. Transfer of conditioned medium that contained detached macrovesicles or co-culture of wild-type target cells with blebbing cells did not stimulate ferroptosis in target cells. Molecular modeling showed that the presence of Hp-phosphatidylethanolamine in the cell membrane promoted its cell ability to be stretched. Together, our data establish that membrane macro-blebbing is characteristic of trophoblast ferroptosis and can serve as a useful marker of this process. Whether or not these blebs are physiologically functional remains to be established.

2.
Proc Natl Acad Sci U S A ; 118(7)2021 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-33531347

RESUMO

Cell-cell adhesions are often subjected to mechanical strains of different rates and magnitudes in normal tissue function. However, the rate-dependent mechanical behavior of individual cell-cell adhesions has not been fully characterized due to the lack of proper experimental techniques and therefore remains elusive. This is particularly true under large strain conditions, which may potentially lead to cell-cell adhesion dissociation and ultimately tissue fracture. In this study, we designed and fabricated a single-cell adhesion micro tensile tester (SCAµTT) using two-photon polymerization and performed displacement-controlled tensile tests of individual pairs of adherent epithelial cells with a mature cell-cell adhesion. Straining the cytoskeleton-cell adhesion complex system reveals a passive shear-thinning viscoelastic behavior and a rate-dependent active stress-relaxation mechanism mediated by cytoskeleton growth. Under low strain rates, stress relaxation mediated by the cytoskeleton can effectively relax junctional stress buildup and prevent adhesion bond rupture. Cadherin bond dissociation also exhibits rate-dependent strengthening, in which increased strain rate results in elevated stress levels at which cadherin bonds fail. This bond dissociation becomes a synchronized catastrophic event that leads to junction fracture at high strain rates. Even at high strain rates, a single cell-cell junction displays a remarkable tensile strength to sustain a strain as much as 200% before complete junction rupture. Collectively, the platform and the biophysical understandings in this study are expected to build a foundation for the mechanistic investigation of the adaptive viscoelasticity of the cell-cell junction.

3.
Proc Natl Acad Sci U S A ; 117(52): 33263-33271, 2020 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-33318201

RESUMO

Gap closure to eliminate physical discontinuities and restore tissue integrity is a fundamental process in normal development and repair of damaged tissues and organs. Here, we demonstrate a nonadhesive gap closure model in which collective cell migration, large-scale actin-network fusion, and purse-string contraction orchestrate to restore the gap. Proliferative pressure drives migrating cells to attach onto the gap front at which a pluricellular actin ring is already assembled. An actin-ring segment switching process then occurs by fusion of actin fibers from the newly attached cells into the actin cable and defusion from the previously lined cells, thereby narrowing the gap. Such actin-cable segment switching occurs favorably at high curvature edges of the gap, yielding size-dependent gap closure. Cellular force microscopies evidence that a persistent rise in the radial component of inward traction force signifies successful actin-cable segment switching. A kinetic model that integrates cell proliferation, actin fiber fusion, and purse-string contraction is formulated to quantitatively account for the gap-closure dynamics. Our data reveal a previously unexplored mechanism in which cells exploit multifaceted strategies in a highly cooperative manner to close nonadhesive gaps.


Assuntos
Actinas/metabolismo , Cicatrização , Animais , Fenômenos Biomecânicos , Adesão Celular , Proliferação de Células , Forma Celular , Cães , Imageamento Tridimensional , Cinética , Células Madin Darby de Rim Canino , Microscopia de Força Atômica , Modelos Biológicos , Fatores de Tempo
4.
Soft Matter ; 15(36): 7203-7210, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31475279

RESUMO

Cancer metastasis has been believed as a genetically programmed process that is commonly marked by biochemical signals. Here using extracellular matrix control of cellular mechanics, we establish that cellular force threshold can also mark in vitro metastatic phenotypic change and malignant transformation in HCT-8 cell colonies. We observe that for prolonged culture time the HCT-8 cell colonies disperse into individual malignant cells, and the metastatic-like dispersion depends on both cell-seeding gel stiffness and colony size. Cellular force microscopies show that gel stiffness and colony size are also two key parameters that modulate cellular forces, suggesting the correlations between the cellular forces and the metastatic phenotypic change. Using our recently developed biophysical model, we construct an extracellular traction phase diagram in the stiffness-size space, filled with experimental data on the colony behavior. From the phase diagram we identify a phase boundary as a traction force threshold above which the metastatic phenotypic transition occurs and below which the cell colonies remain cohesive. Our finding suggests that the traction threshold can be regarded as an effective mechano-marker for the onset of the metastatic-like dispersion and malignant transformation.


Assuntos
Transformação Celular Neoplásica/metabolismo , Células Epiteliais/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Colo/citologia , Matriz Extracelular/metabolismo , Humanos , Mecanotransdução Celular , Modelos Biológicos , Fenótipo , Estresse Mecânico
5.
Sci Adv ; 5(8): eaax0729, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31467978

RESUMO

Cell-cell communication plays a pivotal role in coordination and function of biological systems. Three-dimensional (3D) spheroids provide venues to explore cellular communication for tissue development and drug discovery, as their 3D architecture mimics native in vivo microenvironments. Cellular electrophysiology is a prevalent signaling paradigm for studying electroactive cells. Currently, electrophysiological studies do not provide direct, multisite, simultaneous investigation of tissues in 3D. In this study, 3D self-rolled biosensor arrays (3D-SR-BAs) of either active field-effect transistors or passive microelectrodes were implemented to interface human cardiac spheroids in 3D. The arrays provided continuous and stable multiplexed recordings of field potentials with high sensitivity and spatiotemporal resolution, supported with simultaneous calcium imaging. Our approach enables electrophysiological investigation and monitoring of the complex signal transduction in 3D cellular assemblies toward an organ-on-an-electronic-chip (organ-on-e-chip) platform for tissue maturation investigations and development of drugs for disease treatment, such as arrhythmias.


Assuntos
Técnicas Biossensoriais/métodos , Comunicação Celular , Microeletrodos , Esferoides Celulares/fisiologia , Humanos
6.
Proc Natl Acad Sci U S A ; 115(49): 12359-12364, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30455311

RESUMO

Morphogenesis is a phenomenon by which a wide variety of functional organs are formed in biological systems. In plants, morphogenesis is primarily driven by differential growth of tissues. Much effort has been devoted to identifying the role of genetic and biomolecular pathways in regulating cell division and cell expansion and in influencing shape formation in plant organs. However, general principles dictating how differential growth controls the formation of complex 3D shapes in plant leaves and flower petals remain largely unknown. Through quantitative measurements on live plant organs and detailed finite-element simulations, we show how the morphology of a growing leaf is determined by both the maximum value and the spatial distribution of growth strain. With this understanding, we develop a broad scientific framework for a morphological phase diagram that is capable of rationalizing four configurations commonly found in plant organs: twisting, helical twisting, saddle bending, and edge waving. We demonstrate the robustness of these findings and analyses by recourse to synthetic reproduction of all four configurations using controlled polymerization of a hydrogel. Our study points to potential approaches to innovative geometrical design and actuation in such applications as building architecture, soft robotics and flexible electronics.


Assuntos
Flores/crescimento & desenvolvimento , Orchidaceae/crescimento & desenvolvimento , Desenvolvimento Vegetal , Folhas de Planta/crescimento & desenvolvimento , Modelos Biológicos
7.
Anal Chem ; 90(17): 10340-10349, 2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30088414

RESUMO

We previously reported the finding of a linear correlation between the change of energy dissipation (Δ D) of adhered cells measured with the quartz crystal microbalance with dissipation monitoring (QCM-D) and the level of focal adhesions of the cells. To account for this correlation, we have developed a theoretical framework for assessing the Δ D-response of adhered cells. We rationalized that the mechanical energy of an oscillating QCM-D sensor coupled with a cell monolayer is dissipated through three main processes: the interfacial friction through the dynamic restructuring (formation and rupture) of cell-extracellular matrix (ECM) bonds, the interfacial viscous damping by the liquid trapped between the QCM-D sensor and the basal membrane of the cell layer, and the intracellular viscous damping through the viscous slip between the cytoplasm and stress fibers as well as among stress fibers themselves. Our modeling study shows that the interfacial viscous damping by the trapped liquid is the primary process for energy dissipation during the early stage of the cell adhesion, whereas the dynamic restructuring of cell-ECM bonds becomes more prevalent during the later stage of the cell adhesion. Our modeling study also establishes a positive linear correlation between the Δ D-response and the level of cell adhesion quantified with the number of cell-ECM bonds, which corroborates our previous experimental finding. This correlation with a wide well-defined linear dynamic range provides a much needed theoretical validation of the dissipation monitoring function of the QCM-D as a powerful quantitative analytical tool for cell study.


Assuntos
Adesão Celular , Metabolismo Energético , Técnicas de Microbalança de Cristal de Quartzo , Matriz Extracelular/metabolismo , Modelos Teóricos
8.
Adv Mater ; 30(27): e1707464, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29797358

RESUMO

Targeted delivery of nanoparticle (NP)-based diagnostic and therapeutic agents to malignant cells and tissues has exclusively relied on chemotargeting, wherein NPs are surface-coated with ligands that specifically bind to overexpressed receptors on malignant cells. Here, it is demonstrated that cellular uptake of NPs can also be biased to malignant cells based on the differential mechanical states of cells, enabling mechanotargeting. Owing to mechanotransduction, cell lines (HeLa and HCT-8) cultured on hydrogels of various stiffness are directed into different stress states, measured by cellular force microscopies. In vitro NP delivery reveals that increases in cell stress suppress cellular uptake, counteracting the enhanced uptake that occurs with increases in exposed surface area of spread cells. Upon prolonged culture on stiff hydrogels, cohesive HCT-8 cell colonies undergo metastatic phenotypic change and disperse into individual malignant cells. The metastatic cells are of extremely low stress state and adopt an unspread, 3D morphology, resulting in several-fold higher uptake than the nonmetastatic counterparts. This study opens a new paradigm of harnessing mechanics for the design of future strategies in nanomedicine.


Assuntos
Nanopartículas , Transporte Biológico , Humanos , Mecanotransdução Celular , Microscopia de Força Atômica , Nanomedicina
9.
Small ; 14(12): e1702495, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29430869

RESUMO

Stably transfected cell lines are widely used in drug discovery and biological research to produce recombinant proteins. Generation of these cell lines requires the isolation of multiple clones, using time-consuming dilution methods, to evaluate the expression levels of the gene of interest. A new and efficient method is described for the generation of monoclonal cell lines, without the need for dilution cloning. In this new method, arrays of patterned cell colonies and single cell transfection are employed to deliver a plasmid coding for a reporter gene and conferring resistance to an antibiotic. Using a nanofountain probe electroporation system, probe positioning is achieved through a micromanipulator with sub-micron resolution and resistance-based feedback control. The array of patterned cell colonies allows for rapid selection of numerous stably transfected clonal cell lines located on the same culture well, conferring a significant advantage over slower and labor-intensive traditional methods. In addition to plasmid integration, this methodology can be seamlessly combined with CRISPR/Cas9 gene editing, paving the way for advanced cell engineering.


Assuntos
Sistemas CRISPR-Cas/genética , Eletroporação/métodos , Animais , Linhagem Celular , Edição de Genes/métodos , Humanos , Plasmídeos/genética , Transfecção
10.
Proc Natl Acad Sci U S A ; 115(1): 70-74, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29255037

RESUMO

Many applications in tissue engineering, flexible electronics, and soft robotics call for approaches that are capable of producing complex 3D architectures in soft materials. Here we present a method using molecular self-assembly to generate hydrogel-based 3D architectures that resembles the appealing features of the bottom-up process in morphogenesis of living tissues. Our strategy effectively utilizes the three essential components dictating living tissue morphogenesis to produce complex 3D architectures: modulation of local chemistry, material transport, and mechanics, which can be engineered by controlling the local distribution of polymerization inhibitor (i.e., oxygen), diffusion of monomers/cross-linkers through the porous structures of cross-linked polymer network, and mechanical constraints, respectively. We show that oxygen plays a role in hydrogel polymerization which is mechanistically similar to the role of growth factors in tissue growth, and the continued growth of hydrogel enabled by diffusion of monomers/cross-linkers into the porous hydrogel similar to the mechanisms of tissue growth enabled by material transport. The capability and versatility of our strategy are demonstrated through biomimetics of tissue morphogenesis for both plants and animals, and its application to generate other complex 3D architectures. Our technique opens avenues to studying many growth phenomena found in nature and generating complex 3D structures to benefit diverse applications.


Assuntos
Materiais Biomiméticos , Hidrogéis , Engenharia Tecidual , Tecidos Suporte/química , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/química , Humanos , Hidrogéis/síntese química , Hidrogéis/química , Porosidade
11.
Mol Biol Cell ; 28(23): 3156-3164, 2017 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-28495795

RESUMO

The translation of mechanical forces into biochemical signals plays a central role in guiding normal physiological processes during tissue development and homeostasis. Interfering with this process contributes to cardiovascular disease, cancer progression, and inherited disorders. The actin-based cytoskeleton and its associated adherens junctions are well-established contributors to mechanosensing and transduction machinery; however, the role of the desmosome-intermediate filament (DSM-IF) network is poorly understood in this context. Because a force balance among different cytoskeletal systems is important to maintain normal tissue function, knowing the relative contributions of these structurally integrated systems to cell mechanics is critical. Here we modulated the interaction between DSMs and IFs using mutant forms of desmoplakin, the protein bridging these structures. Using micropillar arrays and atomic force microscopy, we demonstrate that strengthening the DSM-IF interaction increases cell-substrate and cell-cell forces and cell stiffness both in cell pairs and sheets of cells. In contrast, disrupting the interaction leads to a decrease in these forces. These alterations in cell mechanics are abrogated when the actin cytoskeleton is dismantled. These data suggest that the tissue-specific variability in DSM-IF network composition provides an opportunity to differentially regulate tissue mechanics by balancing and tuning forces among cytoskeletal systems.


Assuntos
Desmoplaquinas/metabolismo , Desmoplaquinas/fisiologia , Filamentos Intermediários/metabolismo , Citoesqueleto de Actina/metabolismo , Junções Aderentes/metabolismo , Fenômenos Biomecânicos/fisiologia , Caderinas/metabolismo , Adesão Celular/fisiologia , Citoesqueleto/metabolismo , Desmossomos/metabolismo , Humanos , Filamentos Intermediários/fisiologia
12.
Proc Natl Acad Sci U S A ; 114(11): 2910-2915, 2017 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-28265065

RESUMO

When detergents and phospholipid membranes are dispersed in aqueous solutions, they tend to self-assemble into vesicles of various shapes and sizes by virtue of their hydrophobic and hydrophilic segments. A clearer understanding of such vesiculation processes holds promise for better elucidation of human physiology and disease, and paves the way to improved diagnostics, drug development, and drug delivery. Here we present a detailed analysis of the energetics and thermodynamics of vesiculation by recourse to nonlinear elasticity, taking into account large deformation that may arise during the vesiculation process. The effects of membrane size, spontaneous curvature, and membrane stiffness on vesiculation and vesicle size distribution were investigated, and the critical size for vesicle formation was determined and found to compare favorably with available experimental evidence. Our analysis also showed that the critical membrane size for spontaneous vesiculation was correlated with membrane thickness, and further illustrated how the combined effects of membrane thickness and physical properties influenced the size, shape, and distribution of vesicles. These findings shed light on the formation of physiological extracellular vesicles, such as exosomes. The findings also suggest pathways for manipulating the size, shape, distribution, and physical properties of synthetic vesicles, with potential applications in vesicle physiology, the pathobiology of cancer and other diseases, diagnostics using in vivo liquid biopsy, and drug delivery methods.


Assuntos
Fosfolipídeos/química , Lipossomas Unilamelares/química , Exossomos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Modelos Biológicos , Tamanho da Partícula
13.
Tumour Biol ; 2016 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-27900564

RESUMO

This study examined the effects of RNAi-mediated TUSC3 silencing on radiation-induced autophagy and radiation sensitivity of human lung adenocarcinoma cell line A549 under hypoxic condition. Different CoCl2 concentrations were used to treat A549 cells and establish a CoCl2-induced hypoxic model of A549 cells. MTT and clone formation assays were used to determine the effects of different concentrations of CoCl2 on the growth and proliferation of A549 cells treated by different doses of X-ray irradiation. The siRNA-expressing vector was transfected by liposomes and for silencing of TUSC3. Flow cytometry was used to measure cell cycle changes and apoptosis rate. Real-time quantitative polymerase chain reaction (qRT-PCR) assay was performed to detect the expression of TUSC3 mRNA. Western blotting was applied to detect the changes of TUSC3, LC3, and p62 proteins under different CoCl2 concentrations and after siRNA silencing of TUSC3. The TUSC3 levels in A549 cells increased under hypoxic conditions in a dose-dependent manner (P < 0.05). Hypoxia inhibited the growth and proliferation of A549 cells and promoted apoptosis (P < 0.05). With an increasing dose of X-ray irradiation, A549 cells showed significantly increased growth and proliferation and decreased apoptosis (P < 0.05). After siRNA-TUSC3 was transfected by liposome, the TUSC3 level was substantially inhibited (P < 0.05). Silencing TUSC3 inhibited A549 cell growth and proliferation after radiotherapy under hypoxic condition, promoted apoptosis, increased G0/G1 phase cells, and reduced S phase cells (all P < 0.05). Hypoxia and radiation along with different CoCl2 concentrations could induce cell autophagy, which increased with concentration and dose, while silencing the TUSC3 gene inhibited autophagy (all P < 0.05). RNAi silencing of TUSC3 inhibited growth and proliferation, while enhanced apoptosis and radiation sensitivity of hypoxic A549 lung adenocarcinoma cells.

14.
Proc Natl Acad Sci U S A ; 113(17): 4800-5, 2016 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-27071094

RESUMO

The sexual blood stage of the human malaria parasite Plasmodium falciparum undergoes remarkable biophysical changes as it prepares for transmission to mosquitoes. During maturation, midstage gametocytes show low deformability and sequester in the bone marrow and spleen cords, thus avoiding clearance during passage through splenic sinuses. Mature gametocytes exhibit increased deformability and reappear in the peripheral circulation, allowing uptake by mosquitoes. Here we define the reversible changes in erythrocyte membrane organization that underpin this biomechanical transformation. Atomic force microscopy reveals that the length of the spectrin cross-members and the size of the skeletal meshwork increase in developing gametocytes, then decrease in mature-stage gametocytes. These changes are accompanied by relocation of actin from the erythrocyte membrane to the Maurer's clefts. Fluorescence recovery after photobleaching reveals reversible changes in the level of coupling between the membrane skeleton and the plasma membrane. Treatment of midstage gametocytes with cytochalasin D decreases the vertical coupling and increases their filterability. A computationally efficient coarse-grained model of the erythrocyte membrane reveals that restructuring and constraining the spectrin meshwork can fully account for the observed changes in deformability.


Assuntos
Deformação Eritrocítica , Eritrócitos/ultraestrutura , Estágios do Ciclo de Vida , Microtúbulos/ultraestrutura , Modelos Biológicos , Plasmodium falciparum/ultraestrutura , Actinas/ultraestrutura , Simulação por Computador , Citoesqueleto/ultraestrutura , Espectrina/ultraestrutura
15.
J Biomed Mater Res B Appl Biomater ; 104(3): 488-95, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25939598

RESUMO

Improving targeting efficacy has been a central focus of the studies on nanoparticle (NP)-based drug delivery nanocarriers over the past decades. As cells actively sense and respond to the local physical environments, not only the NP design (e.g., size, shape, ligand density, etc.) but also the cell mechanics (e.g., stiffness, spreading, expressed receptors, etc.) affect the cellular uptake efficiency. While much work has been done to elucidate the roles of NP design for cells seeded on a flat tissue culture surface, how the local physical environments of cells mediate uptake of NPs remains unexplored, despite the widely known effect of local physical environments on cellular responses in vitro and disease states in vivo. Here, we report the active responses of human osteosarcoma cells to fibrous substrate topographies and the subsequent changes in the cellular uptake of NPs. Our experiments demonstrate that surface topography modulates cellular uptake efficacy by mediating cell spreading and membrane mechanics. The findings provide a concrete example of the regulative role of the physical environments of cells on cellular uptake of NPs, therefore advancing the rational design of NPs for enhanced drug delivery in targeted cancer therapy.


Assuntos
Portadores de Fármacos , Nanopartículas/química , Linhagem Celular Tumoral , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacologia , Humanos , Nanopartículas/ultraestrutura , Propriedades de Superfície
16.
Sci Rep ; 5: 10525, 2015 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-26013284

RESUMO

Physical penetration of lipid bilayer membranes presents an alternative pathway for cellular delivery of nanoparticles (NPs) besides endocytosis. NPs delivered through this pathway could reach the cytoplasm, thereby opening the possibility of organelle-specific targeting. Herein we perform dissipative particle dynamics simulations to elucidate the transmembrane penetration mechanisms of multiple NPs. Our simulations demonstrate that NPs' translocation proceeds in a cooperative manner, where the interplay of the quantity and surface chemistry of the NPs regulates the translocation efficiency. For NPs with hydrophilic surfaces, the increase of particle quantity facilitates penetration, while for NPs with partly or totally hydrophobic surfaces, the opposite highly possibly holds. Moreover, a set of interesting cooperative ways, such as aggregation, aggregation-dispersion, and aggregation-dispersion-reaggregation of the NPs, are observed during the penetration process. We find that the penetration behaviors of multiple NPs are mostly dominated by the changes of the NP-membrane force components in the membrane plane direction, in addition to that in the penetration direction, suggesting a different interaction mechanism between the multiple NPs and the membrane compared with the one-NP case. These results provide a fundamental understanding in the underlying mechanisms of cooperative penetration of NPs, and shed light on the NP-based drug and gene delivery.


Assuntos
Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Nanopartículas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Nanopartículas/química
17.
Oncotarget ; 6(12): 9834-53, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25868853

RESUMO

Malignant melanoma, the most deadly form of skin cancer, has a high propensity for metastatic spread and is notoriously chemotherapy-resistant. Cordycepin, the active component of Cordyceps spp., has been identified to have anti-metastatic effect on tumor progression and thus possesses pharmacological and therapeutic potentials. However, the mechanisms of anti-metastatic effects of cordycepin at cellular levels remain elusive. We analyzed the effect of cordycepin on human melanoma miRNA expression profiles by miRNAarray and found that miR-33b was upregulated in highly-metastatic melanoma cell lines following cordycepin exposure. Cordycepin-mediated miR-33b expression was dependent on LXR-RXR heterodimer activation. miR-33b directly binds to HMGA2, Twist1 and ZEB1 3'-UTR to suppress their expression. The negative correlations between miR-33b levels and HMGA2, Twist1 or ZEB1 expression were detected in 72 patient melanoma tissue samples. By targeting HMGA2 and Twist1, miR-33b attenuated melanoma migration and invasiveness upon cordycepin exposure. miR-33b knockdown or ZEB1 overexpression reverted cordycepin-mediated mesenchymal-epithelial transition (MET), triggering the expression of N-cadherin. In spontaneous metastasis models, cordycepin suppressed tumor metastasis without altering primary tumor growth. We showed for the first time that targeting miRNA by cordycepin indicates a new mechanism of cordycepin-induced suppression of tumor metastasis and miR-33b/HMGA2/Twist1/ZEB1 axis plays critical roles in regulating melanoma dissemination.


Assuntos
Desoxiadenosinas/farmacologia , Proteína HMGA2/metabolismo , Proteínas de Homeodomínio/metabolismo , Melanoma/metabolismo , MicroRNAs/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Cutâneas/metabolismo , Fatores de Transcrição/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Regiões 3' não Traduzidas , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Citoesqueleto/metabolismo , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica , Metástase Neoplásica , Transplante de Neoplasias , Cicatrização , Adulto Jovem , Homeobox 1 de Ligação a E-box em Dedo de Zinco
18.
Proc Natl Acad Sci U S A ; 112(19): 6068-73, 2015 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-25918423

RESUMO

During its asexual development within the red blood cell (RBC), Plasmodium falciparum (Pf), the most virulent human malaria parasite, exports proteins that modify the host RBC membrane. The attendant increase in cell stiffness and cytoadherence leads to sequestration of infected RBCs in microvasculature, which enables the parasite to evade the spleen, and leads to organ dysfunction in severe cases of malaria. Despite progress in understanding malaria pathogenesis, the molecular mechanisms responsible for the dramatic loss of deformability of Pf-infected RBCs have remained elusive. By recourse to a coarse-grained (CG) model that captures the molecular structures of Pf-infected RBC membrane, here we show that nanoscale surface protrusions, known as "knobs," introduce multiple stiffening mechanisms through composite strengthening, strain hardening, and knob density-dependent vertical coupling. On one hand, the knobs act as structural strengtheners for the spectrin network; on the other, the presence of knobs results in strain inhomogeneity in the spectrin network with elevated shear strain in the knob-free regions, which, given its strain-hardening property, effectively stiffens the network. From the trophozoite to the schizont stage that ensues within 24-48 h of parasite invasion into the RBC, the rise in the knob density results in the increased number of vertical constraints between the spectrin network and the lipid bilayer, which further stiffens the membrane. The shear moduli of Pf-infected RBCs predicted by the CG model at different stages of parasite maturation are in agreement with experimental results. In addition to providing a fundamental understanding of the stiffening mechanisms of Pf-infected RBCs, our simulation results suggest potential targets for antimalarial therapies.


Assuntos
Membrana Eritrocítica/parasitologia , Eritrócitos/parasitologia , Malária Falciparum/parasitologia , Plasmodium falciparum , Simulação por Computador , Citoesqueleto/química , Contagem de Eritrócitos , Humanos , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Resistência ao Cisalhamento , Estresse Mecânico
19.
Toxicol Lett ; 234(3): 151-61, 2015 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-25725129

RESUMO

Diphenyl difluoroketone (EF24), a curcumin analog, exhibits potent anti-tumor activities by arresting cell cycle and inducing apoptosis. However, the efficacy and modes of action of EF24 on melanoma metastasis remain elusive. In this study, we found that at non-cytotoxic concentrations, EF24 suppressed cell motility and epithelial-to-mesenchymal Transition (EMT) of melanoma cell lines, Lu1205 and A375. EF24 also suppressed HMGA2 expression at mRNA and protein levels. miR-33b directly bound to HMGA2 3' untranslated region (3'-UTR) to suppress its expression as measured by dual-luciferase assay. EF24 increased expression of E-cadherin and decreased STAT3 phosphorylation and expression of the mesenchymal markers, vimentin and N-cadherin. miR-33b inhibition or HMGA2 overexpression reverted EF24-mediated suppression of EMT phenotypes. In addition, EF24 modulated the HMGA2-dependent actin stress fiber formation, focal adhesion assembly and FAK, Src and RhoA activation by targeting miR-33b. Thus, the results suggest that EF24 suppresses melanoma metastasis via upregulating miR-33b and concomitantly reducing HMGA2 expression. The observed activities of EF24 support its further evaluation as an anti-metastatic agent in melanoma therapy.


Assuntos
Antineoplásicos/farmacologia , Compostos de Benzilideno/farmacologia , Movimento Celular/fisiologia , Curcumina/análogos & derivados , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Proteína HMGA2/efeitos dos fármacos , Melanoma/metabolismo , MicroRNAs/efeitos dos fármacos , Piperidonas/farmacologia , Western Blotting , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/fisiologia , Imunofluorescência , Humanos , Melanoma/tratamento farmacológico , Melanoma/fisiopatologia , MicroRNAs/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima/efeitos dos fármacos
20.
Nano Lett ; 13(9): 4546-50, 2013 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-23972158

RESUMO

Nanoparticles (NPs) hold great promises for targeted disease diagnosis and therapy. Despite considerable progress in biomimetic design of NP-bioconjugates, the roles of NP size and shape in endocytosis are still not fully understood. Using an efficient coarse-grained molecular dynamics (CGMD) model, we simulate receptor-mediated endocytosis of NPs of various sizes and shapes. Our simulations demonstrate that both NP size and shape modulate the kinetics of endocytosis. For spherical NPs, there exists an optimal size at which endocytosis takes the shortest time. For a spherocylindrical NP with the initial upright docking position on the membrane plane, endocytosis proceeds through a laying-down-then-standing-up sequence. A free energy analysis reveals that NP size primarily determines whether endocytosis can complete, while NP shape breaks the symmetry of curvature energy landscape and hence dictates the endocytic pathway and the angle of entry. The findings shed light on the rational design of NP-based diagnostic and therapeutic agents with improved cellular targeting.


Assuntos
Endocitose/fisiologia , Nanopartículas , Neoplasias/terapia , Humanos , Cinética , Simulação de Dinâmica Molecular , Nanopartículas/química , Nanopartículas/uso terapêutico , Neoplasias/diagnóstico , Tamanho da Partícula
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