Specific Receptor-ligand Interactions Research Articles

Extracellular vesicle integrins act as a nexus for platelet adhesion in cerebral microvessels

Circulating extracellular vesicles (EVs) regulate signaling pathways via receptor-ligand interactions and content delivery, after attachment or internalization by endothelial cells. However, they originate from diverse cell populations and are heterogeneous in composition. To determine the effects of specific surface molecules, the use of synthetic EV mimetics permits the study of specific EV receptor-ligand interactions. Here, we used endogenous EVs derived from the circulation of rats, as well as ligand-decorated synthetic microparticles (MPs) to examine the role of integrin αvβ3 in platelet adhesion under flow in structurally intact cerebral arteries. At an intraluminal pressure of 50 mmHg and flow rate of 10 µl/min, platelets were delivered to the artery lumen and imaged with whole-field fluorescent microscopy. Under basal conditions very few platelets bound to the endothelium. However, adhesion events were markedly increased following the introduction of arginine-glycine-aspartate (RGD)-labelled synthetic MPs or endogenously-derived EVs from experimental stroke animals carrying excess RGD proteins, including vitronectin, CD40-ligand and thrombospondin-1. These data, which were generated in a dynamic and physiologically relevant system, demonstrate the importance of vesicle-carried RGD ligands in platelet adherence to the cerebrovascular endothelium and highlight the ability of synthetic EVs to isolate and identify key components of the molecular handshake between EVs and their targets.

Abstract 4500: Mechanisms of tumor-associated myeloid cells in modulating host immune microenvironment and metastatic progression

Abstract The immune checkpoint inhibitor blockade takes advantage of the specific receptor-ligand interactions between immune and tumor cells. However, the immunosuppressive microenvironment has been largely ignored, which likely contribute to low efficacy and resistance in immune therapies. Our studies demonstrate that TGF-β signaling in tumor-associated myeloid cells isessential in systemic immune suppression and metastatic progression. Using mouse models of breast cancer metastasis, we further uncover that miR130a and 145 are critical in regulating TGF-β signaling in myeloid cells. miR130a and 145 are down-regulated in these myeloid cells, leading to increased TGF-β RII. Ectopic expression of miR-130a and miR-145 in the myeloid cells decrease tumor metastasis. This is mediated through a downregulation of type 2 cytokines in myeloid cells and an increase in IFNgamma-producing cytotoxic CD8 T lymphocytes. Lastly, miR-130a and miR-145 mimics improve systemic anti-tumor immunity and inhibit metastasis in preclinical mouse models. Another important mechanism in myeloid-mediated immune suppression involves a decreased expression of Lamin A/C, nuclear lamina proteins that prominently interact with heterochromatin. Lamin A/C knockout in myeloid cells leads to H3K4me3-mediated upregulation of Gfi1 and C/EBPϵ,critical transcription factors responsible for granulocytic lineage differentiation. In addition, loss of Lamin A/C attenuates antigen-presentation and compromises host anti-tumor immunity. These mechanistic understandings provide options to reprogram tumor-associated myeloid cells by altering the cytokine milieu and metastatic microenvironment, thus enhancing host antitumor immunity. Citation Format: Hiroki Ishii, Christine Hollander, Tim Greten, Li Yang. Mechanisms of tumor-associated myeloid cells in modulating host immune microenvironment and metastatic progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4500.

Transferrin Functionization Elevates Transcytosis of Nanogranules across Epithelium by Triggering Polarity-Associated Transport Flow and Positive Cellular Feedback Loop.

Overcoming the epithelial barriers to enhance drug transport is a focused topic for gastrointestinal, intratracheal, intranasal, vaginal, and intrauterine delivery. Nanomedicines with targeting functionization promote such a process owing to specific ligand-receptor interaction. However, compared to the cell uptake of targeting nanotherapies, currently few studies concentrate on their transcytosis including endocytosis for "in" and exocytosis for "out". In fact, the cellular regulatory mechanism for these pathways as well as the principle of ligand's effect on the transcytosis are almost ignored. Here, we fabricated transferrin (Tf) functionalized nanogranules (Tf-NG) as the nanomedicine model and confirmed the difference in polar distributions of Tf receptors (TfRs) between two epithelium models (bipolarity for Caco-2 and unipolarity for MDCK cells). Compared to the nonspecific reference, Tf-conjugation boosted the endocytosis by different pathways in two cell models and transformed the intracellular route of Tf-NG in both cells differently, affecting exocytosis, recycling, and degradation but not the secretion pathway. Only bipolar cells could establish a complete transport flow from "in" to "out", leading to the enhanced transcytosis of Tf-NG. Importantly, epithelia could make responses to Tf-NG transcytosis. Based on the quantitative proteomics, the intracellular trafficking of Tf-NG altered the protein expression profiles, in which the endocytosis- and transcytosis-related proteins were specifically upregulated. Particularly, only bipolar cells could positively feed back to such trafficking via accelerating the subsequent Tf-NG transcytosis. Here, all the cell transport of Tf-NG was polarity associated. In summary, Tf modification elevated the transcytosis of Tf-NG across the epithelium by triggering the polarity-associated transport flow and positive cell feedback loop. These findings provided an insight into the targeting nanodelivery for efficient transport through epithelial barriers.

Demystifying a hexuronic acid ligand that recognizes Toxoplasma gondii and blocks its invasion into host cells.

Toxoplasma gondii is a ubiquitous eukaryotic pathogen responsible for toxoplasmosis in humans and animals. This parasite is an obligate intracellular pathogen and actively invades susceptible host cells, a process which is mediated by specific receptor-ligand interactions. Here, we have identified an unnatural 2,4-disulfated d-glucuronic acid (Di-S-GlcA), a hexuronic acid composed of heparin/heparan sulfate, as a potential carbohydrate ligand that can selectively bind to T. gondii parasites. More importantly, the gelatin conjugated Di-S-GlcA multivalent probe displayed strong inhibition of parasite entry into host cells. These results open perspective for the future use of Di-S-GlcA epitopes in biomedical applications against toxoplasmosis.

Radiosensitization of breast cancer cells using AS1411 aptamer-conjugated gold nanoparticles

Introduction: A main choice for cancer treatment is radiotherapy. But, the radiotherapy disadvantage is damages caused by radiation given to normal tissues/organs surrounding cancer. One way to avoid this is via increasing radiosensitization of cancer cells. Gold nanoparticles (GNPs) have shown sensitizing effect on cancer cells by enhancing their absorbed dose. Unlike earlier delivery techniques developed for nanotherapeutics, active targeting can achieve specific effect and higher uptake of GNPs in tumors while leave healthy cells untouched and consequently improve the therapeutic index. To achieve active targeting, GNPs should be equipped with functional ligands which can recognize and adhere to the receptors of cancer cells. Aptamers are small DNA-molecule/RNA-fragments with high specificity and affinity towards target molecules. AS1411 aptamer can specifically bind to over-expressed nucleolin on the membrane of tumor cells including breast cancer. This aptamer is capable to enter cancer cells through specific ligand–receptor interaction. Greater uptake of GNPs by cells may induce increased radiation effects. Cancer stem cells are a small population of cells within a tumor capable for self-renewal and differentiation into various cell types. Materials and Methods: We hypothesized that conjugation of GNPs with AS1411 (AS1411/GNPs) could increase GNPs-mediated radiosensitization in breast cancer cells. We hypothesized that AS1411/GNPs would radiosensitize breast cancer stem/progenitor cells grown to three- dimensional (3D) mammospheres. Cytotoxicity studies of the GNPs and AS1411/GNPs were done on two different cancer cell lines of MCF-7 and MDA-MB-231 with MTT assay. Atomic absorption spectroscopy (AAS) confirmed the cellular uptake of particles. Radiosensitizing effect of GNPs and AS1411/GNPs on MDA-MB 231 and MCF- 7 cells assessed by clonogenic assay. Results: Clonogenic survival data revealed that AS1411/GNPs at 12.5 mg/L results in radiosensitization of breast cancer cells. Mammosphere of MCF-7 was more resistant than their monolayer counterparts. Conclusion: The combination of the sensitizing GNPs with the AS14411 aptamer can be regarded for improved treatment of breast cancer cells especially for the mammosphere MCF-7 cancer cells mimicking cancerous tumors.

In silico and in vitro analysis of boAP3d1 protein interaction with bovine leukaemia virus gp51.

The envelope glycoprotein 51 (gp51) is essential for bovine leukaemia virus (BLV) entry to bovine B-lymphocytes. Although the bovine adaptor protein 3 complex subunit delta-1 (boAP3D1) has been proposed as the potential receptor, the specific ligand-receptor interaction has not yet been completely defined and boAP3D1 receptor and gp51 3D structures have not been determined. This study was thus aimed at a functional annotation of boAP3D1 cellular adaptor protein and BLV gp51 and, proposing a reliable model for gp51-AP3D1 interaction using bioinformatics tools. The boAP3D1 receptor interaction patterns were calculated based on models of boAP3D1 receptor and gp51 complexes’ 3D structures, which were constructed using homology techniques and data-driven docking strategy. The results showed that the participation of 6 key amino acids (aa) on gp51 (Asn170, Trp127, His115, Ala97, Ser98 and Glu128) and 4 aa on AP3D1 (Lys925, Asp807, Asp695 and Arg800) was highly probable in the interaction between gp51 and BLVR domains. Three gp51 recombinant peptides were expressed and purified to validate these results: the complete domain (rgp51), the N-terminal portion (rNgp51) and the C-terminal fragment (rCgp51); and binding assays to Madin-Darby bovine kidney (MDBK) cells were then carried out with each recombinant. It was found that rNgp51 preferentially bound to MDBK cells, suggesting this domain’s functional role during invasion. The rNgp51-MDBK cell interaction was sensitive to trypsin (98% reduction) and chymotrypsin treatment (80% reduction). These results highlighted that the N-terminal portion of gp51 interacted in vitro with the AP3D1 receptor and provides a plausible in silico interaction model.

To Bind or Not to Bind: Unravelling GPCR Polypharmacology

Interaction of a single drug with multiple targets through "polypharmacology" is increasingly recognized as necessary for treatment of complex diseases, such as schizophrenia. G protein-coupled receptors (GPCRs) are major medicinal targets, and understanding the structural basis of both GPCR drug selectivity and promiscuity could provide novel avenues for drug development.

Galectin-1 Influences Breast Cancer Cell Adhesion to E-selectin Via Ligand Intermediaries.

Invasion of other tissues during bloodborne metastasis in part requires adhesion of cancer cells to vascular endothelium by specific fluid shear-dependent receptor-ligand interactions. This study investigates the hypothesis that the adhesion is mediated by ligands shared between endothelial E-selectin and Galectin-1 (Gal-1), both of which are upregulated during inflammation and cancer. Flow chamber adhesion and dynamic biochemical tissue analysis (DBTA) assays were used to evaluate whether Gal-1 modulates E-selectin adhesive interactions of breast cancer cells and tissues under dynamic flow conditions, while immunocytochemistry, immunohistochemistry, western blotting, and fluorescence anisotropy were used to study molecular interactions under static conditions. Dynamic adhesion assays revealed a shear-dependent binding interaction between Gal-1hFc treated breast cancer cells and tissues and E-selectin-coated beads, causing~300% binding increase of the beads compared to negative controls. Immunocyto- and immunohistochemical analyses showed that Gal-1 and E-selectin fluorescent signals colocalized on cells and tissues at~75% for each assay. Immunoprecipitation and Western blotting of Mac-2BP from breast cancer cell lysates revealed that Gal-1 and E-selectin share Mac-2BP as a ligand, while fluorescence anisotropy and circulating tumor cell model systems exhibited competitive or antagonistic binding between Gal-1 and E-selectin for shared ligands, including Mac-2BP. Furthermore, Mac-2BP functional blockade inhibited the effects of Gal-1 on E-selectin binding. In summary, this investigation reveals a shear-dependent interaction between E-selectin and Gal-1 that may be due to intermediation by a similar or shared ligand(s), including Mac-2BP, which may provide a rational basis for development of novel diagnostics or therapeutics for breast cancer.

Synacton and individual activity of synthetic and natural corticotropins.

Short endogenous peptides represent one of the most important constituents of the mammalian body's general regulatory system. Some synthesized analogs and modified natural peptides (eg, corticotropins) also show high biological activity. Nevertheless, the mechanism of action of regulatory peptides remains unclear. To explain the effects of peptides of intermolecular processes, the hypothesis that a synactonal mechanism underlies the action of regulatory peptides, exemplified by the heptapeptide Semax, has been proposed. Thus, in the total pool of Semax metabolites, which includes the cleavage products of the parental molecule, we can distinguish the functional core, represented by the major metabolic products-peptides HFPGP and PGP. These peptides have their own binding sites with similar although differing characteristics. Together with Semax, they constitute a single complex of bioregulators acting in a certain sequence and in interaction, ie, synacton. It can be assumed that the diverse clinically significant effects of the drug Semax are determined by its synacton. Specific interactions between some tritium-labeled peptides (basic constituents of the Semax synacton) and plasma membranes of neurons have been characterized. Only a few peptides of the Semax synacton showed competitive activity for the Semax binding sites. Fragments comprising 5 amino acid residues (EHFPG and HFPGP) showed the highest competitive activity. We also characterized the processes of specific ligand-receptor interactions of some tritium-labeled corticotropins ([3 H-Pro]MEHFPGP, [3 H-Pro]HFPGP, and [3 H-Pro]PGP) by applying mathematical discriminative models (Scatchard, Hill, Bjerrum, and Lineweaver-Burk plots). So the intermolecular interactions of these peptides with plasma membranes of neuronal brain targets are probably not limited by specific binding at orthosteric sites. The effect of peptides that act in the synacton considerably extends the regulatory potential of the initial molecule.

Leishmania donovani Utilize Sialic Acids for Binding and Phagocytosis in the Macrophages through Selective Utilization of Siglecs and Impair the Innate Immune Arm

BackgroundLeishmania donovani, belonging to a unicellular protozoan parasite, display the differential level of linkage-specific sialic acids on their surface. Sialic acids binding immunoglobulin-like lectins (siglecs) are a class of membrane-bound receptors present in the haematopoetic cell lineages interact with the linkage-specific sialic acids. Here we aimed to explore the utilization of sialic acids by Leishmania donovani for siglec-mediated binding, phagocytosis, modulation of innate immune response and signaling pathways for establishment of successful infection in the host.Methodology/Principle FindingsWe have found enhanced binding of high sialic acids containing virulent strains (AG83+Sias) with siglec-1 and siglec-5 present on macrophages compared to sialidase treated AG83+Sias (AG83-Sias) and low sialic acids-containing avirulent strain (UR6) by flow cytometry. This specific receptor-ligand interaction between sialic acids and siglecs were further confirmed by confocal microscopy. Sialic acids-siglec-1-mediated interaction of AG83+Sias with macrophages induced enhanced phagocytosis. Additionally, sialic acids-siglec-5 interaction demonstrated reduced ROS, NO generation and Th2 dominant cytokine response upon infection with AG83+Sias in contrast to AG83-Sias and UR6. Sialic acids-siglecs binding also facilitated multiplication of intracellular amastigotes. Moreover, AG83+Sias induced sialic acids-siglec-5-mediated upregulation of host phosphatase SHP-1. Such sialic acids-siglec interaction was responsible for further downregulation of MAPKs (p38, ERK and JNK) and PI3K/Akt pathways followed by the reduced translocation of p65 subunit of NF-κβ to the nucleus from cytosol in the downstream signaling pathways. This sequence of events was reversed in AG83-Sias and UR6-infected macrophages. Besides, siglec-knockdown macrophages also showed the reversal of AG83+Sias infection-induced effector functions and downstream signaling events.Conclusions/SignificancesTaken together, this study demonstrated that virulent parasite (AG83+Sias) establish a unique sialic acids-mediated binding and subsequent phagocytosis in the host cell through the selective exploitation of siglec-1. Additionally, sialic acids-siglec-5 interaction altered the downstream signaling pathways which contributed impairment of immune effector functions of macrophages. To the best of our knowledge, this is a comprehensive report describing sialic acids-siglec interactions and their role in facilitating uptake of the virulent parasite within the host.

Design of cholesterol arabinogalactan anchored liposomes for asialoglycoprotein receptor mediated targeting to hepatocellular carcinoma: In silico modeling, in vitro and in vivo evaluation

We have developed active targeting liposomes to deliver anticancer agents to ASGPR which will contribute to effective treatment of hepatocellular carcinoma. Active targeting is achieved through polymeric ligands on the liposome surface. The liposomes were prepared using reverse phase evaporation method and doxorubicin hydrocholoride, a model drug, was loaded using the ammonium sulphate gradient method. Liposomes loaded with DOX were found to have a particle size of 200nm with more than 90% entrapment efficiency. Systems were observed to release the drug in a sustained manner in acidic pH in vitro. Liposomes containing targeting ligands possessed greater and selective toxicity to ASGPR positive HepG2 cell lines due to specific ligand receptor interaction. Bio-distribution studies revealed that liposomes were concentrated in the liver even after 3h of administration, thus providing conclusive evidence of targeting potential for formulated nanosystems. Tumor regression studies indicated greater tumor suppression with targeted liposomes thereby establishing superiority of the liposomal system. In this work, we used a novel methodology to guide the determination of the optimal composition of the targeting liposomes: molecular dynamics (MD) simulation that aided our understanding of the behaviour of the ligand within the bilayer. This can be seen as a demonstration of the utility of this methodology as a rational design tool for active targeting liposome formulation.

Molecular Basis of the Receptor Interactions of Polysialic Acid (polySia), polySia Mimetics, and Sulfated Polysaccharides.

Polysialic acid (polySia) and polySia glycomimetic molecules support nerve cell regeneration, differentiation, and neuronal plasticity. With a combination of biophysical and biochemical methods, as well as data mining and molecular modeling techniques, it is possible to correlate specific ligand-receptor interactions with biochemical processes and in vivo studies that focus on the potential therapeutic impact of polySia, polySia glycomimetics, and sulfated polysaccharides in neuronal diseases. With this strategy, the receptor interactions of polySia and polySia mimetics can be understood on a submolecular level. As the HNK-1 glycan also enhances neuronal functions, we tested whether similar sulfated oligo- and polysaccharides from seaweed could be suitable, in addition to polySia, for finding potential new routes into patient care focusing on an improved cure for various neuronal diseases. The knowledge obtained here on the structural interplay between polySia or sulfated polysaccharides and their receptors can be exploited to develop new drugs and application routes for the treatment of neurological diseases and dysfunctions.

Regulating the Rate of Molecular Self‐Assembly for Targeting Cancer Cells

AbstractBesides tight and specific ligand–receptor interactions, the rate regulation of the formation of molecular assemblies is one of fundamental features of cells. But the latter receives little exploration for developing anticancer therapeutics. Here we show that a simple molecular design of the substrates of phosphatases—tailoring the number of phosphates on peptidic substrates—is able to regulate the rate of molecular self‐assembly of the enzyme reaction product. Such a rate regulation allows selective inhibition of osteosarcoma cells over hepatocytes, which promises to target cancer cells in a specific organ. Moreover, our result reveals that the direct measurement of the rate of the self‐assembly in a cell‐based assay provides precise assessment of the cell targeting capability of self‐assembly. This work, as the first report establishing rate regulation of a multiple‐step process to inhibit cells selectively, illustrates a fundamentally new approach for controlling the fate of cells.

Cellular Adhesion: Evaluating the Effect of Receptor-Ligand Chemistries, Distribution of Receptors, and Spread Versus Spherical Cell Geometry

The initial stage of cellular adhesion is similar to a functionalized nanocarrier binding to a cell membrane through specific receptor-ligand interactions. Therefore, the computational models developed for nanocarrier binding can be used to investigate cell-substrate interactions. In this work, we use a Monte Carlo based free energy model to determine the binding affinities of a cell interacting with a functionalized substrate. In particular, we focus on how the chemical variations of the cell surface receptors (e.g., integrins) mediate the adhesive interactions and thereby affect the binding affinity. Our multiscale model explicitly accounts for (i) a mixture of specific and non-specific receptor-ligand interactions, (ii) flexural motion of the ligands, (iii) thermal motion of the cell, and (iv) feedback between molecular adhesion and membrane dynamics. The receptor-ligand interactions are first characterized using conventional docking studies and the interaction energy landscape is determined from all atom umbrella sampling simulations. For simplicity, we model the dynamics of the cell membrane using a harmonic potential that mimics the undulations of the membrane. We first study how the underlying molecular interactions, composition of receptor, and size and shape of the cell manifest as effective association constants. We also show that binding affinities, computed for cells whose dimensions are less than 1 micron, follow an asymptotic scaling behavior that can be used to predict the binding affinities of typical mammalian cells with larger dimension (> 1 micron) with minimal computational costs.

Live Imaging Studies of Invasion Phenotypes of Erythrocytes by P. Falciparum

Severe malaria by Plasmodium falciparum is a leading cause of death in many developing countries. In the blood stage of the disease, merozoite invasion of erythrocytes is a repetitive intraerythrocytic cycle which takes place over a ∼ 48-hour period. Infection of healthy blood cells occurs only within a few minutes after parasite egression, and is a critical point in the progression of the disease1. Furthermore, this process involving complex receptor-ligand binding processes. To understand specific receptor-ligand interactions, we investigate morphology and kinetics during P. Falciparum invasion with various antibodies filmed by video microscopy, building on [2]. We quantify invasion phenotypes such as efficiency, timescales and pathogen/host adhesion strength. This study aims to understand mechanism of malaria invasion and aid the design of drugs or vaccines that could block this stage of the parasite's life cycle.In parallel to static cultures we develop microfluidic devices to achieve high throughput of merozosites under controlled flows. Infected red blood cells (IRBCs) are delivered into a channel containing cage-like “nests”, which block these relatively stiff cells. The IRBS then continue maturation, delivering a stream of parasites. Non infected RBCs are delivered from another input, and pass unhindered through the nests, to a long serpentine channel where they co-flow with the egressed parasites. This setup allows the observation of parasite invasion in flow, to identify the changes in invasion under variant flow and with increased throughput than static cultures.

Binding Forces of Single αMβ2 Integrin-Fibrinogen Interactions on Living Cells

Single Cell Force Spectroscopy (SCFS) can be used to measure the maximum adhesion force between cells and a surface, but determining the nature of discrete interactions within SCFS data can be challenging. We present a method for quantifying specific ligand-receptor interactions on the single molecule level on living cells using atomic force microscopy (AFM) based SCFS experiments. SCFS data from HEK 293 cells expressing αMβ2 leukocyte integrin (HEK Mac-1) and wild-type HEK 293 (HEK WT) cells on surfaces coated with fibrinogen were analyzed to identify specific “rupture events.” High force load ruptures (> 0.2 pN/nm) imply a connection of the integrin with the underlying actin cortex of the cell, while low force load (< 0.2 pN/nm) ruptures result from the formation of a membrane tether. For highly adhesive fibrinogen surfaces, we found 41% of all rupture events to have a high force load for HEK Mac-1 cells compared to only 9% of rupture events having a high force load for HEK WT data of the same surface. The high force load events in the HEK Mac-1 data showed a median rupture force of 55 pN, whereas the median rupture force of the HEK WT high force load events was 29 pN. After adding monoclonal antibody directed against the αM subunit of the integrin, HEK Mac-1 cells showed similar rupture force values to that of the HEK WT. This analysis demonstrates the ability to quantify specific integrin-ligand interactions within SCFS data.

Targeting Cancer using Polymeric Nanoparticle mediated Combination Chemotherapy.

Cancer forms exhibiting poor prognosis have been extensively researched for therapeutic solutions. One of the conventional modes of treatment, chemotherapy shows inadequacy in its methodology due to imminent side-effects and acquired drug-resistance by cancer cells. However, advancements in nanotechnology have opened new frontiers to significantly alleviate collateral damage caused by current treatments via innovative delivery techniques, eliminating pitfalls encountered in conventional treatments. Properties like reduced drug-clearance and increased dose efficacy by the enhanced permeability and retention effect deem nanoparticles suitable for this application. Optimization of size, surface charge and surface modifications have provided nanoparticles with stealth properties capable of evading immune responses, thus deeming them as excellent carriers of chemotherapeutic agents. Biocompatible and biodegradable forms of polymers enhance the bioavailability of chemotherapeutic agents, and permit a sustained and time-dependent release of drugs which is a characteristic of their composition, thereby providing a controlled therapeutic approach. Studies conducted in vitro and animal models have also demonstrated a synergism in cytotoxicity given the mechanism of action of anticancer drugs when administered in combination providing promising results. Combination therapy has also shown implications in overcoming multiple-drug resistance, which can however be subdued by the adaptable nature of tumor microenvironment. Surface modifications with targeting moieties can therefore feasibly increase nanoparticle uptake by specific receptor-ligand interactions, increasing dose efficacy which can seemingly overcome drug-resistance. This article reviews recent trends and investigations in employing polymeric nanoparticles for effectively delivering combination chemotherapy, and modifications in delivery parameters enhancing dose efficacy, thus validating the potential in this approach for anticancer treatment.

A20-Binding Inhibitor of NF-κB Activation 1 is a Physiologic Inhibitor of NF-κB: A Molecular Switch for Inflammation and Autoimmunity.

The transcription factor NF-κB comprises a family of transcription factors critical for inflammatory signaling, and innate and adaptive immunity.1 NF-κB regulates the transcription of genes involved in immuno-inflammatory responses, cell survival, cell cycle progression, and cell adhesion. Activation of NF-κB by specific ligand-receptor interactions induces the expression of pro-inflammatory mediators, which orchestrate the recruitment of immune cells, induce the inflammatory response, and cause tissue injury.2,3 In this way, excessive NF-κB activity is considered harmful and a pathogenic factor in the chronic inflammation of numerous autoimmune disorders, such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), type 1 diabetes, multiple sclerosis, and inflammatory bowel disease.4 Association between overactive NF-κB and autoimmunity emphasizes the importance of tightly controlled NF-κB signaling. One protein that regulates NF-κB signaling and maintains immune homeostasis is the A20 binding inhibitor of NF-κB1 (ABIN1).

Abstract 342: Breast cancer adhesion cascade is influenced by Galectin-1 and E-selectin receptor-ligand interactions

Abstract Invasion of other tissues during bloodborne metastasis requires the adhesion of cancer cells to the endothelium by specific fluid shear-dependent receptor-ligand interactions. It is hypothesized that ligands are shared between the endothelial adhesion molecule known as E-selectin, a transmembrane glycoprotein, and Galectin-1 (Gal-1), a secretory dimer protein, both of which are upregulated during inflammation and cancer. Our previous data strongly indicate that this shear-dependent interaction involves the same or closely related cancer cell-expressed ligands, such as the glycoprotein Mac-2BP. Initial investigation using immuno-staining methods to locate E-selectin and Gal-1 ligands and determine relative proximity to one another revealed that E-selectin and Gal-1 ligands on ZR-75-1 breast cancer cells and breast cancer tissue microarrays had a high percentage of colocalization, as high as 75% in some cases. These results allude to the possibility that these reactive molecules are in close proximity or are in fact the same molecule. Subsequently, E-selectin microspheres were perfused over Gal-1-treated ZR-75-1 cell monolayers and breast cancer tissue microarrays to determine the functional relationship, if any, between Gal-1 and E-selectin ligands during adhesion from flow. Flow assay data show that Gal-1 treatment enhances E-selectin microsphere interactions to a breast cancer monolayer in a shear-dependent manner, with a shear stress of 0.5 dynes/cm2 having an almost 300% increase in binding compared to the untreated monolayer. This amount of binding at a physiological shear stress gives credence to the hypothesis that Gal-1 influences E-selectin receptor-ligand interactions via the same or similar ligands during breast cancer cell adhesion. Dynamic biochemical tissue analysis (DBTA), a novel flow-based tissue interrogation technique developed in our lab, was then implemented to test human invasive ductal breast carcinoma tissue sections. Similar to ZR-75-1 cell line flow assay data, a 142% increase in shear-dependent binding interactions between the E-selectin microspheres and the Gal-1 treated breast cancer tissue sections at 0.5 dynes/cm2 shear stress was observed. Colocalization of reactive signals and increased shear-dependent flow interactions indicate that metastatic binding in breast cancer is aided through E-selectin to Gal-1 interaction via a shared ligand intermediate. Ascertaining the mechanism behind this interaction would lead to a better understanding of the circulating breast cancer cells’ adhesion cascade during metastasis. Citation Format: Nathan M. Reynolds, Claire R. Hall, Sean E. Thomas, Monica M. Burdick. Breast cancer adhesion cascade is influenced by Galectin-1 and E-selectin receptor-ligand interactions. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 342. doi:10.1158/1538-7445.AM2015-342

Structure-Function Basis of Attenuated Inverse Agonism of Angiotensin II Type 1 Receptor Blockers for Active-State Angiotensin II Type 1 Receptor

Ligand-independent signaling by the angiotensin II type 1 receptor (AT1R) can be activated in clinical settings by mechanical stretch and autoantibodies as well as receptor mutations. Transition of the AT1R to the activated state is known to lower inverse agonistic efficacy of clinically used AT1R blockers (ARBs). The structure-function basis for reduced efficacy of inverse agonists is a fundamental aspect that has been understudied not only in relation to the AT1R but also regarding other homologous receptors. Here, we demonstrate that the active-state transition in the AT1R indeed attenuates an inverse agonistic effect of four biphenyl-tetrazole ARBs through changes in specific ligand-receptor interactions. In the ground state, tight interactions of four ARBs with a set of residues (Ser109(TM3), Phe182(ECL2), Gln257(TM6), Tyr292(TM7), and Asn295(TM7)) results in potent inverse agonism. In the activated state, the ARB-AT1R interactions shift to a different set of residues (Val108(TM3), Ser109(TM3), Ala163(TM4), Phe182(ECL2), Lys199(TM5), Tyr292(TM7), and Asn295(TM7)), resulting in attenuated inverse agonism. Interestingly, V108I, A163T, N295A, and F182A mutations in the activated state of the AT1R shift the functional response to the ARB binding toward agonism, but in the ground state the same mutations cause inverse agonism. Our data show that the second extracellular loop is an important regulator of the functional states of the AT1R. Our findings suggest that the quest for discovering novel ARBs, and improving current ARBs, fundamentally depends on the knowledge of the unique sets of residues that mediate inverse agonistic potency in the two states of the AT1R.