ABSTRACT
Small molecules that interfere with microtubule dynamics, such as Taxol and the Vinca alkaloids, are widely used in cell biology research and as clinical anticancer drugs. However, their activity cannot be restricted to specific target cells, which also causes severe side effects in chemotherapy. Here, we introduce the photostatins, inhibitors that can be switched on and off in vivo by visible light, to optically control microtubule dynamics. Photostatins modulate microtubule dynamics with a subsecond response time and control mitosis in living organisms with single-cell spatial precision. In longer-term applications in cell culture, photostatins are up to 250 times more cytotoxic when switched on with blue light than when kept in the dark. Therefore, photostatins are both valuable tools for cell biology, and are promising as a new class of precision chemotherapeutics whose toxicity may be spatiotemporally constrained using light.
Subject(s)
Antimitotic Agents/chemistry , Cell Death , Microtubules/drug effects , Mitosis , Stilbenes/chemistry , Animals , Antimitotic Agents/toxicity , Cell Line, Tumor , Cytoskeleton/chemistry , Humans , Light , Mice , Polymerization , Stilbenes/toxicityABSTRACT
Notch signaling is critical for many developmental and disease-related processes. It is widely accepted that Notch has a mechanotransduction module that regulates receptor cleavage. However, the role of biomechanical properties of the cellular environment in Notch signaling in general is still poorly understood. During angiogenesis, differentiation of endothelial cells into tip and stalk cells is regulated by Notch signaling, and remodeling of the extracellular matrix occurs. We investigated the influence of substrate stiffness on the Notch signaling pathway in endothelial cells. Using stiffness-tuned polydimethylsiloxane (PDMS) substrates, we show that activity of the Notch signaling pathway inversely correlates with a physiologically relevant range of substrate stiffness (i.e. increased Notch signaling activity on softer substrates). Trans-endocytosis of the Notch extracellular domain, but not the overall endocytosis, is regulated by substrate stiffness, and integrin cell-matrix connections are both stiffness dependent and influenced by Notch signaling. We conclude that mechanotransduction of Notch activation is modulated by substrate stiffness, highlighting the role of substrate rigidity as an important cue for signaling. This might have implications in pathological situations associated with stiffening of the extracellular matrix, such as tumor growth.
Subject(s)
Endothelial Cells , Mechanotransduction, Cellular , Endothelial Cells/metabolism , Signal Transduction/physiology , Cell Differentiation , Extracellular Matrix/metabolism , Receptors, Notch/genetics , Receptors, Notch/metabolism , Calcium-Binding Proteins/metabolism , Neovascularization, Physiologic/physiologyABSTRACT
Lagunamide A is a biologically active natural product with a yet unidentified molecular mode of action. Cellular studies revealed that lagunamide A is a potent inhibitor of cancer cell proliferation, promotes apoptosis and causes mitochondrial dysfunction. To decipher the cellular mechanism responsible for these effects, we utilized thermal protein profiling (TPP) and identified EYA3 as a stabilized protein in cells upon lagunamide A treatment. EYA3, involved in the DNA damage repair process, was functionally investigated via siRNA based knockdown studies and corresponding effects of lagunamide A on DNA repair were confirmed. Furthermore, we showed that lagunamide A sensitized tumor cells to treatment with the drug doxorubicin highlighting a putative therapeutic strategy.
Subject(s)
Antineoplastic Agents , Apoptosis , Cell Proliferation , DNA Damage , DNA Repair , Proteome , Humans , Apoptosis/drug effects , Cell Proliferation/drug effects , DNA Damage/drug effects , DNA Repair/drug effects , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Proteome/drug effects , Proteome/metabolism , Proteome/analysis , Cell Line, Tumor , Doxorubicin/pharmacologyABSTRACT
Cell dispersion from a confined area is fundamental in a number of biological processes, including cancer metastasis. To date, a quantitative understanding of the interplay of single-cell motility, cell proliferation, and intercellular contacts remains elusive. In particular, the role of E- and N-cadherin junctions, central components of intercellular contacts, is still controversial. Combining theoretical modeling with in vitro observations, we investigate the collective spreading behavior of colonies of human cancer cells (T24). The spreading of these colonies is driven by stochastic single-cell migration with frequent transient cell-cell contacts. We find that inhibition of E- and N-cadherin junctions decreases colony spreading and average spreading velocities, without affecting the strength of correlations in spreading velocities of neighboring cells. Based on a biophysical simulation model for cell migration, we show that the behavioral changes upon disruption of these junctions can be explained by reduced repulsive excluded volume interactions between cells. This suggests that in cancer cell migration, cadherin-based intercellular contacts sharpen cell boundaries leading to repulsive rather than cohesive interactions between cells, thereby promoting efficient cell spreading during collective migration.
Subject(s)
Cadherins , Neoplasms , Cell Adhesion , Cell Communication , Cell Movement , Cell Proliferation , HumansABSTRACT
Since the first report on a yeast three-hybrid system, several approaches have successfully utilized different setups for discovering targets of small molecule drugs. Compared to broadly applied MS based target identification approaches, the yeast three-hybrid system represents a complementary method that allows for the straightforward identification of direct protein binders of selected small molecules. One major drawback of this system, however, is that the drug has to be taken up by the yeast cells in sufficient concentrations. Here, we report the establishment of a yeast three-hybrid screen in the deletion strain ABC9Δ, which is characterized by being highly permeable to small molecules. We used this system to screen for protein binding partners of ethinylestradiol, a widely used drug mainly for contraception and hormone replacement therapy. We identified procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2 or lysyl hydroxylase, LH2) as a novel direct target and were able to confirm the interaction identified with the yeast three-hybrid system by a complementary method, affinity chromatography, to prove the validity of the hit. Furthermore, we provide evidence for an interaction between the drug and PLOD2 in vitro and in cellulo.
Subject(s)
Ethinyl Estradiol , Saccharomyces cerevisiae , Ethinyl Estradiol/pharmacology , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/metabolism , Protein Binding , Saccharomyces cerevisiae/metabolism , Two-Hybrid System TechniquesABSTRACT
Chlorophyll and heme are among the "pigments of life", tetrapyrrolic structures, without which life on Earth would not be possible. Their catabolites, the phyllobilins and the bilins, respectively, share not only structural features, but also a similar story: Long considered waste products of detoxification processes, important bioactivities for both classes have now been demonstrated. For phyllobilins, however, research on physiological roles is sparse. Here, we introduce actin, the major component of the cytoskeleton, as the first discovered target of phyllobilins and as a novel target of bilins. We demonstrate the inhibition of actin dynamics in vitro and effects on actin and related processes in cancer cells. A direct interaction with G-actin is shown by in silico studies and confirmed by affinity chromatography. Our findings open a new chapter in bioactivities of tetrapyrroles-especially phyllobilins-for which they form the basis for broad implications in plant science, ecology, and physiology.
Subject(s)
Actins/antagonists & inhibitors , Chlorophyll/chemistry , Heme/chemistry , Pigments, Biological/pharmacology , Tetrapyrroles/pharmacology , Actins/metabolism , Humans , Pigments, Biological/chemistry , Tetrapyrroles/chemistryABSTRACT
Cell-permeable photoswitchable small molecules, termed optojasps, are introduced to optically control the dynamics of the actin cytoskeleton and cellular functions that depend on it. These light-dependent effectors were designed from the F-actin-stabilizing marine depsipeptide jasplakinolide by functionalizing them with azobenzene photoswitches. As demonstrated, optojasps can be employed to control cell viability, cell motility, and cytoskeletal signaling with the high spatial and temporal resolution that light affords. Optojasps can be expected to find applications in diverse areas of cell biological research. They may also provide a template for photopharmacology targeting the ubiquitous actin cytoskeleton with precision control in the micrometer range.
Subject(s)
Actins/chemistry , Azo Compounds/chemistry , Depsipeptides/chemistry , Small Molecule Libraries/chemistry , Azo Compounds/chemical synthesis , Molecular Conformation , Photochemical Processes , Small Molecule Libraries/chemical synthesisABSTRACT
Developmental processes, such as angiogenesis, are associated with a constant remodeling of the actin cytoskeleton in response to different mechanical stimuli. The mechanosensitive transcription factors MRTF-A (MKL1) and YAP (also known as YAP1) are important mediators of this challenging adaptation process. However, it is as yet unknown whether both pathways respond in an identical or in a divergent manner to a given microenvironmental guidance cue. Here, we use a micropatterning approach to dissect single aspects of cellular behavior in a spatiotemporally controllable setting. Using the exemplary process of angiogenesis, we show that cell-cell contacts and adhesive surface area are shared regulatory parameters of MRTF and YAP on rigid 2D surfaces. By analyzing MRTF and YAP under laminar flow conditions and during cell migration on dumbbell-shaped microstructures, we demonstrate that they exhibit different translocation kinetics. In conclusion, our work promotes the application of micropatterning techniques as a cell biological tool to study mechanosensitive signaling in the context of angiogenesis.
Subject(s)
Actins/metabolism , Blood Vessels/metabolism , Cytological Techniques/methods , Human Umbilical Vein Endothelial Cells/chemistry , Human Umbilical Vein Endothelial Cells/metabolism , Mechanotransduction, Cellular , Actins/chemistry , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Blood Vessels/chemistry , Blood Vessels/growth & development , Humans , Kinetics , Mice , Mice, Inbred C57BL , Neovascularization, Physiologic , Phosphoproteins/genetics , Phosphoproteins/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors , YAP-Signaling ProteinsABSTRACT
Therapeutic options for patients with advanced-stage hepatocellular carcinoma (HCC) are very limited. The only approved first-line treatment is the multi-tyrosine kinase inhibitor sorafenib, which shows low response rates and severe side effects. In particular, the compensatory activation of growth factor receptors leads to chemoresistance and limits the clinical impact of sorafenib. However, combination approaches to improve sorafenib have failed. Here we investigate the inhibition of cyclin-dependent kinase 5 (Cdk5) as a promising combination strategy to improve sorafenib response in HCC. Combination of sorafenib with Cdk5 inhibition (genetic knockdown by short hairpin RNA or CRISPR/Cas9 and pharmacologic inhibition) synergistically impaired HCC progression in vitro and in vivo by inhibiting both tumor cell proliferation and migration. Importantly, these effects were mediated by a mechanism for Cdk5: A liquid chromatography-tandem mass spectrometry-based proteomic approach revealed that Cdk5 inhibition interferes with intracellular trafficking, a process crucial for cellular homeostasis and growth factor receptor signaling. Cdk5 inhibition resulted in an accumulation of enlarged vesicles and respective cargos in the perinuclear region, considerably impairing the extent and quality of growth factor receptor signaling. Thereby, Cdk5 inhibition offers a comprehensive approach to globally disturb growth factor receptor signaling that is superior to specific inhibition of individual growth factor receptors. Conclusion: Cdk5 inhibition represents an effective approach to improve sorafenib response and to prevent sorafenib treatment escape in HCC. Notably, Cdk5 is an addressable target frequently overexpressed in HCC, and with Dinaciclib, a clinically tested Cdk5 inhibitor is readily available. Thus, our study provides evidence for clinically evaluating the combination of sorafenib and Dinaciclib to improve the therapeutic situation for patients with advanced-stage HCC.
Subject(s)
Carcinoma, Hepatocellular/drug therapy , Cyclin-Dependent Kinase 5/antagonists & inhibitors , Liver Neoplasms/drug therapy , Protein Kinase Inhibitors/therapeutic use , Sorafenib/therapeutic use , Animals , Female , Humans , Mice , Treatment Outcome , Tumor Cells, CulturedABSTRACT
In this letter, we report a series of five new RGD-containing cyclic peptides as potent inhibitors to αvß3 integrin protein. We have incorporated various unnatural lipophilic amino acids into the cyclic RGD framework of cilengitide, which is selective for αvß3 integrin. All the newly synthesized cyclic peptides were evaluated in vitrosolid phase binding assay and investigated for their bindingbehaviourtowards integrin subtypes. All the cyclic peptides were synthesized in excellent yield following solution-phase coupling strategy. The cyclic RGD peptides 1a-e exhibited IC50 of 9.9, 5.5, 72, 11 and 3.3 nM, respectively, towardsαvß3 integrin protein. This finding offers further opportunities for the introduction unusual amino acids into the cyclic peptide framework of cilengitide.
Subject(s)
Integrin alphaVbeta3/antagonists & inhibitors , Peptides, Cyclic/pharmacology , Dose-Response Relationship, Drug , Humans , Integrin alphaVbeta3/metabolism , Molecular Structure , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/chemistry , Structure-Activity RelationshipABSTRACT
Combination chemotherapy has proven to be a favorable strategy to treat acute leukemia. However, the introduction of novel compounds remains challenging and is hindered by a lack of understanding of their mechanistic interactions with established drugs. In the present study, we demonstrate a highly increased response of various acute leukemia cell lines, drug-resistant cells and patient-derived xenograft cells by combining the recently introduced protein disulfide isomerase inhibitor PS89 with cytostatics. In leukemic cells, a proteomics-based target fishing approach revealed that PS89 affects a whole network of endoplasmic reticulum homeostasis proteins. We elucidate that the strong induction of apoptosis in combination with cytostatics is orchestrated by the PS89 target B-cell receptor-associated protein 31, which transduces apoptosis signals at the endoplasmic reticulum -mitochondria interface. Activation of caspase-8 and cleavage of B-cell receptor-associated protein 31 stimulate a pro-apoptotic crosstalk including release of calcium from the endoplasmic reticulum and an increase in the levels of reactive oxygen species resulting in amplification of mitochondrial apoptosis. The findings of this study promote PS89 as a novel chemosensitizing agent for the treatment of acute leukemia and uncovers that targeting the endoplasmic reticulum - mitochondrial network of cell death is a promising approach in combination therapy.
Subject(s)
Cytostatic Agents/pharmacology , Endoplasmic Reticulum/metabolism , Leukemia/metabolism , Mitochondria/metabolism , Signal Transduction/drug effects , Animals , Apoptosis/drug effects , Caspase 8/metabolism , Cell Line, Tumor , Disease Models, Animal , Drug Resistance, Neoplasm/drug effects , Humans , Leukemia/drug therapy , Leukemia/pathology , Mice , Models, Biological , Proteome , Proteomics/methods , Xenograft Model Antitumor AssaysABSTRACT
OBJECTIVE: Ischemia-reperfusion (I/R) injury significantly contributes to organ dysfunction and failure after myocardial infarction, stroke, and transplantation. In addition to its established role in the fibrinolytic system, plasminogen activator inhibitor-1 has recently been implicated in the pathogenesis of I/R injury. The underlying mechanisms remain largely obscure. APPROACH AND RESULTS: Using different in vivo microscopy techniques as well as ex vivo analyses and in vitro assays, we identified that plasminogen activator inhibitor-1 rapidly accumulates on microvascular endothelial cells on I/R enabling this protease inhibitor to exhibit previously unrecognized functional properties by inducing an increase in the affinity of ß2 integrins in intravascularly rolling neutrophils. These events are mediated through low-density lipoprotein receptor-related protein-1 and mitogen-activated protein kinase-dependent signaling pathways that initiate intravascular adherence of these immune cells to the microvascular endothelium. Subsequent to this process, extravasating neutrophils disrupt endothelial junctions and promote the postischemic microvascular leakage. Conversely, deficiency of plasminogen activator inhibitor-1 effectively reversed leukocyte infiltration, microvascular dysfunction, and tissue injury on experimental I/R without exhibiting side effects on microvascular hemostasis. CONCLUSIONS: Our experimental data provide novel insights into the nonfibrinolytic properties of the fibrinolytic system and emphasize plasminogen activator inhibitor-1 as a promising target for the prevention and treatment of I/R injury.
Subject(s)
Abdominal Muscles/blood supply , Liver/blood supply , Microvessels/metabolism , Neutrophil Infiltration , Neutrophils/metabolism , Plasminogen Activator Inhibitor 1/metabolism , Reperfusion Injury/metabolism , Abdominal Muscles/metabolism , Abdominal Muscles/pathology , Animals , CD18 Antigens/metabolism , Capillary Permeability , Cell Line , Disease Models, Animal , Humans , Kinetics , Leukocyte Rolling , Liver/metabolism , Liver/pathology , Low Density Lipoprotein Receptor-Related Protein-1 , Male , Mice, Inbred C57BL , Mice, Knockout , Microvessels/pathology , Neutrophil Activation , Neutrophils/transplantation , Plasminogen Activator Inhibitor 1/deficiency , Plasminogen Activator Inhibitor 1/genetics , Protein Conformation , Receptors, LDL/metabolism , Reperfusion Injury/pathology , Signal Transduction , Tumor Suppressor Proteins/metabolismABSTRACT
Actin is a protein of central importance for many cellular key processes. It is regulated by local interactions with a large number of actin binding proteins (ABPs). Various compounds are known to either increase or decrease the polymerization dynamics of actin. However, no actin binding compound has been developed for clinical applications yet because of selectivity issues. We provide a crystal structure of the natural product chivosazole A (ChivoA) bound to actin and show that-in addition to inhibiting nucleation, polymerization, and severing of F-actin filaments-it selectively modulates binding of ABPs to G-actin: Although unphysiological actin dimers are induced by ChivoA, interaction with gelsolin, profilin, cofilin, and thymosin-ß4 is inhibited. Moreover, ChivoA causes transcriptional effects differing from latrunculin B, an actin binder with a different binding site. Our data show that ChivoA and related compounds could serve as scaffolds for the development of actin binding molecules selectively targeting specific actin functions.
Subject(s)
Actins/metabolism , Macrolides/pharmacology , Binding Sites , Crystallography, X-Ray , Human Umbilical Vein Endothelial Cells , Humans , Molecular Structure , Protein BindingABSTRACT
Actin has emerged as a versatile regulator of gene transcription. Cytoplasmatic actin regulates mechanosensitive-signaling pathways such as MRTF-SRF and Hippo-YAP/TAZ. In the nucleus, both polymerized and monomeric actin directly interfere with transcription-associated molecular machineries. Natural actin-binding compounds are frequently used tools to study actin-related processes in cell biology. However, their influence on transcriptional regulation and intranuclear actin polymerization is poorly understood to date. Here, we analyze the effects of two representative actin-binding compounds, Miuraenamide A (polymerizing properties) and Latrunculin B (depolymerizing properties), on transcriptional regulation in primary cells. We find that actin stabilizing and destabilizing compounds inversely shift nuclear actin levels without a direct influence on polymerization state and intranuclear aspects of transcriptional regulation. Furthermore, we identify Miuraenamide A as a potent inducer of G-actin-dependent SRF target gene expression. In contrast, the F-actin-regulated Hippo-YAP/TAZ axis remains largely unaffected by compound-induced actin aggregation. This is due to the inability of AMOTp130 to bind to the amorphous actin aggregates resulting from treatment with miuraenamide. We conclude that actin-binding compounds predominantly regulate transcription via their influence on cytoplasmatic G-actin levels, while transcriptional processes relying on intranuclear actin polymerization or functional F-actin networks are not targeted by these compounds at tolerable doses.
Subject(s)
Actins/drug effects , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Depsipeptides/pharmacology , Gene Expression Regulation/drug effects , Thiazolidines/pharmacology , Transcription, Genetic/drug effects , Actins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Nucleus/metabolism , Cells, Cultured , Cytoplasm/drug effects , Cytoplasm/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Mice , NIH 3T3 Cells , Trans-Activators/metabolismABSTRACT
BACKGROUND: Although altered membrane physiology has been discussed within the context of cancer, targeting membrane characteristics by drugs being an attractive therapeutic strategy has received little attention so far. METHODS: Various acetyl-CoA carboxylase 1 (ACC1), and fatty acid synthase (FASN) inhibitors (like Soraphen A and Cerulenin) as well as genetic knockdown approaches were employed to study the effects of disturbed phospholipid composition on membrane properties and its functional impact on cancer progression. By using state-of-the-art methodologies such as LC-MS/MS, optical tweezers measurements of giant plasma membrane vesicles and fluorescence recovery after photobleaching analysis, membrane characteristics were examined. Confocal laser scanning microscopy, proximity ligation assays, immunoblotting as well as migration, invasion and proliferation experiments unravelled the functional relevance of membrane properties in vitro and in vivo. RESULTS: By disturbing the deformability and lateral fluidity of cellular membranes, the dimerisation, localisation and recycling of cancer-relevant transmembrane receptors is compromised. Consequently, impaired activation of growth factor receptor signalling cascades results in abrogated tumour growth and metastasis in different in vitro and in vivo models. CONCLUSIONS: This study highlights the field of membrane properties as a promising druggable cellular target representing an innovative strategy for development of anti-cancer agents.
Subject(s)
Acetyl-CoA Carboxylase/genetics , Enzyme Inhibitors/administration & dosage , Fatty Acid Synthase, Type I/genetics , Lipogenesis/drug effects , Neoplasms/drug therapy , Acetyl-CoA Carboxylase/antagonists & inhibitors , Cell Line, Tumor , Cell Membrane/drug effects , Cell Movement/drug effects , Cell Proliferation , Cerulenin/administration & dosage , Cerulenin/pharmacology , Enzyme Inhibitors/pharmacology , Fatty Acid Synthase, Type I/antagonists & inhibitors , Gene Knockdown Techniques , Humans , Macrolides/administration & dosage , Macrolides/pharmacology , Membrane Fluidity/drug effects , Molecular Targeted Therapy , Neoplasm Invasiveness , Neoplasms/metabolism , Phospholipids/analysis , Photobleaching , Xenograft Model Antitumor AssaysABSTRACT
RATIONALE: Angiogenesis and vessel integrity depend on the adhesion of endothelial cells (ECs) to the extracellular matrix and to adjacent ECs. The focal adhesion protein α-parvin (α-pv) is essential for vascular development. However, the role of α-pv in ECs in vivo is not known. OBJECTIVE: To determine the function of α-pv in ECs during vascular development in vivo and the underlying mechanisms. METHODS AND RESULTS: We deleted the α-pv gene specifically in ECs of mice to study its role in angiogenesis and vascular development. Here, we show that endothelial-specific deletion of α-pv in mice results in late embryonic lethality associated with hemorrhages and reduced vascular density. Postnatal-induced EC-specific deletion of α-pv leads to retinal hypovascularization because of reduced vessel sprouting and excessive vessel regression. In the absence of α-pv, blood vessels display impaired VE-cadherin junction morphology. In vitro, α-pv-deficient ECs show reduced stable adherens junctions, decreased monolayer formation, and impaired motility, associated with reduced formation of integrin-mediated cell-extracellular matrix adhesion structures and an altered actin cytoskeleton. CONCLUSIONS: Endothelial α-pv is essential for vessel sprouting and for vessel stability.
Subject(s)
Adherens Junctions/ultrastructure , Blood Vessels/embryology , Endothelial Cells/cytology , Endothelium, Vascular/physiology , Microfilament Proteins/physiology , Neovascularization, Physiologic/physiology , Adherens Junctions/physiology , Animals , Antigens, CD/analysis , Blood Vessels/growth & development , Cadherins/analysis , Cell Movement , Cell Shape , Cells, Cultured , Cytoskeleton/ultrastructure , Endothelial Cells/metabolism , Endothelium, Vascular/ultrastructure , Extracellular Matrix/ultrastructure , Female , Genes, Lethal , Human Umbilical Vein Endothelial Cells , Male , Mice , Mice, Transgenic , Microfilament Proteins/deficiency , Microfilament Proteins/genetics , Neovascularization, Physiologic/genetics , Pseudopodia/physiology , Pseudopodia/ultrastructure , RNA Interference , RNA, Small Interfering/pharmacology , Retinal Vessels/pathologyABSTRACT
Here, we report novel lipo-oligoaminoamide nanoformulations for targeted intracellular protein delivery. Formulations are generated by first bioreversibly conjugating a sequence-defined amphiphilic lipo-oligomer 728 to the cargo protein via disulfide bonds, followed by formulation of the formed 728-SS-protein conjugate with different helper lipids in various compositions. The triblock oligoaminoamide 728 contains cysteines for reversible covalent protein conjugation and cross-link-stabilization of formed nanoparticles, polyethylene glycol (PEG) for shielding, and providing a hydrophilic domain, eight cationizable succinoyl tetraethylene pentamine (Stp) repeats for endosomal buffering and escape into the cytosol, and a tetra-oleic acid block for hydrophobic stabilization. The added helper lipids are supposed to enhance serum stability of the nanoparticles and provide targeting by lipid-anchored folic acid (FA)-PEG. The optimized protein nanoparticles, including 728, DOPS, cholesterol, DMPE-PEG2000, and the FA-PEG conjugated lipid 1042, presented a high colloidal stability without significant size increase in 72 h. Using cytotoxic ribonuclease A (RNase A) as cargo protein, FA-728-DOPS-DMPE-RNase A nanoformulation could be identified with highest potency of targeted RNase A-mediated folate-receptor-positive KB carcinoma cell killing among all tested formulations, resulting in 85% KB cell killing at a low concentration of 2 µM. These approximately 50 nm sized nanoparticles induced superior 70% KB cell killing even in the presence of 20% serum. Efficient targeted cytosolic delivery by coformulation with helper lipids was also demonstrated by FA-728-DOPS-DMPE-nlsEGFP nanoformulation using enhanced green fluorescent protein (EGFP) as cargo. Furthermore, partial nlsEGFP was imported into the nuclei of KB cells, validating effective endosomal escape, and following nuclear transport mediated by nuclear localization signal on nlsEGFP. As demonstrated, the screening and optimization of nanoformulations with helper lipids and coformulation agents is considered to be an important and rational next step in the development of intracellular biopharmaceuticals, following initial protein conjugate synthesis.
Subject(s)
Drug Delivery Systems/methods , Nanoparticles/chemistry , Phosphatidylethanolamines , Polyethylene Glycols , Ribonuclease, Pancreatic , Animals , Cell Line, Tumor , Humans , Mice , Phosphatidylethanolamines/chemistry , Phosphatidylethanolamines/pharmacokinetics , Phosphatidylethanolamines/pharmacology , Polyethylene Glycols/chemistry , Polyethylene Glycols/pharmacokinetics , Polyethylene Glycols/pharmacology , Ribonuclease, Pancreatic/chemistry , Ribonuclease, Pancreatic/pharmacokinetics , Ribonuclease, Pancreatic/pharmacologyABSTRACT
OBJECTIVE: Cell-matrix interactions are crucial for regulating cellular activities, such as migration. This is of special importance for morphogenic processes, such as angiogenesis (the development of new blood vessels). Most of our understanding of cell migration relies on 2-dimensional (2D) experiments. However, the awareness that 3D settings might elicit different results has increased. Knowledge about endothelial cell (EC) behavior in 3D environments and the influence of matrix composition on EC migration, in particular, is still limited. APPROACH AND RESULTS: We characterize the migration of single ECs through 2 structurally different hydrogels: spongy Matrigel and fibrillar collagen I. Our observations reveal an elongated migration phenotype in Matrigel and a rounded phenotype with pronounced cell blebs (blebs >2 µm) in collagen I, which have not previously been described in ECs. Directed migration seems to depend on Rac1 and Cdc42 in collagen, but not in Matrigel (shown using appropriate pharmacological inhibitors). By applying anti-integrin antibodies and supplementing laminin in collagen gels, we identify laminin as the main determinant of the elongated phenotype. Laminin seems to induce a morphological switch between modes of migration. As an in situ proof of principle, we performed live imaging of EC migration during vascular growth in a murine retina in the absence and presence of anti-integrin antibodies. CONCLUSIONS: We show that, surprisingly, ECs can evade the pharmacological inhibition of central signaling pathways involved in migration (contractility, small GTPases, and proteolysis) by shifting gears between modes of migration. This finding indicates an unexpected contextual plasticity of EC behavior.
Subject(s)
Chemotaxis , Collagen Type I/metabolism , Collagen/metabolism , Extracellular Matrix/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Laminin/metabolism , Proteoglycans/metabolism , Animals , Cell Shape , Cells, Cultured , Cellular Microenvironment , Drug Combinations , Elastic Modulus , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Humans , Hydrogels , Mice, Transgenic , Microscopy, Video , Phenotype , Protein Binding , Proteolysis , Retinal Neovascularization/metabolism , Retinal Neovascularization/physiopathology , Retinal Vessels/metabolism , Signal Transduction , Time Factors , Time-Lapse Imaging , Transfection , cdc42 GTP-Binding Protein/metabolism , rac1 GTP-Binding Protein/metabolismABSTRACT
The hawthorn (Crataegus spp.) extract WS 1442 is used against mild forms of chronic heart failure. This disease is associated with endothelial barrier dysfunction and edema formation. We have recently shown that WS 1442 protects against this dysfunction by a dual mechanism: it both promotes endothelial barrier integrity by activation of a barrier-enhancing pathway (cortactin activation) and inhibits endothelial hyperpermeability by blocking a barrier disruptive pathway (calcium signaling). In this study, we aimed to identify the bioactive compounds responsible for these actions by using a bioactivity-guided fractionation approach. From the four fractions generated from WS 1442 by successive elution with water, 95â% ethanol, methanol, and 70â% acetone, only the water fraction was inactive, whereas the other three triggered a reduction of endothelial hyperpermeability. Analyses of intracellular calcium levels and cortactin phosphorylation were used as readouts to estimate the bioactivity of subfractions and isolated compounds. Interestingly, only the ethanolic fraction interfered with the calcium signaling, whereas only the methanolic fraction led to an activation of cortactin. Thus, the dual mode of action of WS 1442 could be clearly assigned to two distinct fractions. Although the identification of the calcium-active substance(s) was not successful, we could exclude an involvement of phenolic compounds. Cortactin activation, however, could be clearly attributed to oligomeric procyanidins with a distinct degree of polymerization. Taken together, our study provides the first approach to identify the active constituents of WS 1442 that address different cellular pathways leading to the inhibition of endothelial barrier dysfunction.
Subject(s)
Edema/prevention & control , Flavonoids/pharmacology , Plant Extracts/pharmacology , Calcium/metabolism , Cells, Cultured , Chemical Fractionation , Crataegus/chemistry , Endothelium, Vascular/drug effects , Flavonoids/chemistry , Human Umbilical Vein Endothelial Cells , Humans , Plant Extracts/chemistryABSTRACT
For biomedical applications of nanoconstructs, it is a general prerequisite to efficiently reach the desired target site. In this regard, it is crucial to determine the spatiotemporal distribution of nanomaterials at the microscopic tissue level. Therefore, the effect of different surface modifications on the distribution of microinjected quantum dots (QDs) in mouse skeletal muscle tissue has been investigated. In vivo real-time fluorescence microscopy and particle tracking reveal that carboxyl QDs preferentially attach to components of the extracellular matrix (ECM), whereas QDs coated with polyethylene glycol (PEG) show little interaction with tissue constituents. Transmission electron microscopy elucidates that carboxyl QDs adhere to collagen fibers as well as basement membranes, a type of ECM located on the basolateral side of blood vessel walls. Moreover, carboxyl QDs have been found in endothelial junctions as well as in caveolae of endothelial cells, enabling them to translocate into the vessel lumen. The in vivo QD distribution is confirmed by in vitro experiments. The data suggest that ECM components act as a selective barrier depending on QD surface modification. For future biomedical applications, such as targeting of blood vessel walls, the findings of this study offer design criteria for nanoconstructs that meet the requirements of the respective application.