Search

Ethnopharmacological relevance Shenqi Fuzheng (SQ) is a renowned traditional Chinese medicine extracted from Radix Codonopsis and Radix Astragali. Although SQ is widely used to treat myocardial ischemia-reperfusion (I/R) injury, the molecular mechanisms supporting its clinical application remain elusive. Aim of the study The purpose of current study was to understand its cardioprotective effects at the molecular level using network pharmacology approach. Materials and method In an I/R injury animal model, the beneficial pharmacological activities of SQ were confirmed by decreased infarct range observed on drug treated rats versus control group. Additionally, several serum biochemical indicators were in concord with this observation. Subsequently, a microarray experiment was performed to reveal the influence on injured heart at the gene expression level by this TCM injection. We then proposed a network analysis algorithm NTRA to discover the key nodes based on both disease network structure and transcriptomics. Using NRIODN, a method developed by our group previously, the holistic changes on the gene network induced by for I/R injury and SQ treatment were evaluated. Results Pathway enrichment analysis of highly ranked genes by NTRA showed that PPAR and apoptosis pathways were highly related to I/R injury. Finally, western blot results showed increased level of the PPARα and BAX protein in the heart after injection treatment which confirmed the hypothesis. Conclusion In conclusion, our results suggest that SQ injection exerts protective effect against myocardial ischemia-reperfusion injury through multiple pathways, including myocardial energy metabolism improvement, cell adhesion inhibition, inflammatory reaction perturbation, myocardial apoptosis reduction and ventricular remodeling avoidance.

Sickle cell-related vaso-occlusive crises (VOCs) are among the primary clinical manifestations of sickle cell disease (SCD) and are associated with many acute and chronic complications that lead to significant morbidity and mortality. VOCs are caused by the adhesion of leukocytes and sickle erythrocytes to the endothelium, which results in vascular obstruction and tissue ischemia. By blocking the P-selectin- PSGL-1 (P-selectin glycoprotein ligand 1) mediated cell adhesion, crizanlizumab, a recently FDA approved humanized IgG2 anti-P-selectin antibody, reduced the frequency of VOCs in SCD patients and established the proof of principle for this approach (Ataga KI et al., N Engl J Med, 2017). Inclacumab is a novel, fully human IgG4 monoclonal antibody that selectively targets P-selectin and has safely demonstrated sustained anti-cell adhesion effects in over 700 participants including healthy volunteers and patients with cardiovascular disease (Schmitt C et al., J Cardiovasc Pharmacol. 2015; Tardif JC et al., J Am Coll Cardiol, 2013; Morrison M et al., Eur J Clin Pharmacol, 2015; Kling D et al., Thromb Res, 2013). A crystal structure of inclacumab and P-selectin reveals that inclacumab directly binds to an epitope in the PSGL-1 binding region on P-selectin and thus competitively inhibits P-selectin and its ligand interaction. In contrast, crizanlizumab binds to a more distant epitope to the PSGL-1 binding site on P-selectin. To further elucidate differences between the two antibodies, we characterized inclacumab and crizanlizumab in a series of in vitro functional assays including ligand binding affinity, competitive ligand binding by surface plasmon resonance (SPR), P-selectin mediated cell-based adhesion assay and cell-cell interaction with human whole blood samples. In vitro, inclacumab binds to human P-selectin with high affinity and potently suppresses the interaction of P-selectin with its main ligand PSGL-1. Both antibodies exhibited similar binding affinities to P-selectin (KD of 9.9 and 9.1 nM for inclacumab and crizanlizumab, respectively) and comparable potencies at preventing a PSGL-1 mimetic peptide from binding P-selectin (IC50 of 1.9 and 2.2 µg/mL for inclacumab and crizanlizumab, respectively) or blocking the adhesion of PSGL-1 expressing cells to an immobilized P-selectin (IC50 = 430 ng/mL for inclacumab and IC50 = 453 ng/mL for crizanlizumab). However, inclacumab demonstrated greater maximal platelet-leukocyte cell adhesion inhibition in response to thrombin receptor activating peptide (TRAP) in blood samples from both healthy volunteers and subjects with SCD in an in vitro efficacy assay (see figure). Inclacumab is differentiated from crizanlizumab as a fully human monoclonal antibody that directly blocks the PSGL-1 binding region of P-selectin and shows greater maximal inhibition of cell-cell interactions in vitro. At doses up to 20 mg/kg Q4W, which previous clinical trials have shown to be safe and well-tolerated, inclacumab has much greater drug exposure than the approved dose of crizanlizumab (5 mg/kg W0/W2/Q4W) (Ataga KI et al., N Engl J Med, 2017; Schmitt C et al., J Cardiovasc Pharmacol. 2015; Tardif JC et al., J Am Coll Cardiol, 2013). A single dose of inclacumab 20 mg/kg demonstrated full PLA inhibition for ≥84 days in healthy volunteers (Morrison M et al., Eur J Clin Pharmacol, 2015; Kling D et al., Thromb Res, 2013). Inclacumab may allow for a substantially longer and therefore more convenient dosing interval as compared with crizanlizumab. In aggregate, these data suggest that inclacumab has the potential to be a best-in-class P-selectin inhibitor to reduce VOCs in sickle cell disease. Clinical studies of inclacumab in patients with SCD are planned for the 1st half of 2021. Disclosures Geng: Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mihaila:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Yuan:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Strutt:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Benz:Roche Pharmaceuticals: Current Employment. Tang:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mayer:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Oksenberg:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Highlights • Cell adhesion assay is a simple method to identify the receptor-binding motif of the SARS-CoV spike. • Synthetic peptides can be used to narrow the receptor-binding motif. • The 436–445 amino acids are the minimum receptor-binding motif of the SARS-CoV spike. • Cell adhesion assay can be developed as a high throughput system by using a 96-well MTT assay., Emerging life threatening pathogens such as severe acute aspiratory syndrome-coronavirus (SARS-CoV), avian-origin influenzas H7N9, and the Middle East respiratory syndrome coronavirus (MERS-CoV) have caused a high case-fatality rate and psychological effects on society and the economy. Therefore, a simple, rapid, and safe method to investigate a therapeutic approach against these pathogens is required. In this study, a simple, quick, and safe cell adhesion inhibition assay was developed to determine the potential cellular binding site on the SARS-CoV spike protein. Various synthetic peptides covering the potential binding site helped to minimize further the binding motif to 10–25 residues. Following analyses, 2 peptides spanning the 436–445 and 437–461 amino acids of the spike protein were identified as peptide inhibitor or peptide vaccine candidates against SARS-CoV.

In recent years, several research groups proposed new peptidomimetic antagonists of integrins αvβ3, α5β1, αIIbβ3, αvβ6, αvβ5, etc. based on retro sequences of the classic integrin-binding motif RGD. The retro strategy is still largely ignored for the non-RGD-binding α4β1 integrin. Herein we present the first examples of retro sequences for targeting this integrin, composed of Asp or isoAsp equipped with an aromatic cap at the N-terminus, (S)-pyrrolidine-3-carboxylic acid (β2-Pro) as a constrained core, and the amino variant (AMPUMP) of the well-known α4-targeting diphenylurea MPUPA. We discuss α4β1 receptor affinity (SPA), cell adhesion assays, stability in mouse serum, and conformational analysis. For their significant ability to inhibit cell adhesion and remarkable stability, the retro-peptide mimetics BnCO-Asp-β-Pro-AMPUMP (3) and BnCO-isoAsp-β-Pro-AMPUMP (4) represent promising candidates for designing small molecules as potential anti-inflammatory agents.

Macrovipera lebetina obtusa (MLO) snakes presented by several sub-species, which are characterized by a very specific combination of components. Along with common enzymes characteristic of Vipera species, MLO venom contains unique components specific for “obtusa” sub-species, such as disintegrin obtustatin. MLO venom contents metalloproteinases, phospholipase A2, serine proteases, L-alfa amino acid oxidase, few kinds of disintegrins and some other active agents. The cluster of MLO venom components, such as metalloproteinases, obtustatin, C-type lectins and few others are known as cell adhesion inhibition molecules, which are breaking up integrins and cadherins or bind to them. Therefore, the adhesion affecting properties of MLO venom and its action on cell binding in tissue culture is of great interest. We studied effect of venom of MLO living in Armenia on adhesion and metabolic activity of myocardial cells [cardiomyocytes (CM) and cardiac fibroblasts (CF)] and identification of major effector components of MLO venom. Attachment properties of both CM and CF were affected by MLO venom in a dose-dependent manner. Time exposure also has an effect on CMs and CFs attachment to the substrate and to cell-to-cell contact. Longer exposures times result in higher detachment even at the same MLO venom concentrations. Interestingly, both cell types demonstrated increase in metabolic activity upon exposure to non-lethal doses of crude venom. Analysis of individual components of MLO venom resulted improved adhesion of both CM and CF upon inhibition of metalloproteinases by EDTA-Na2 chelating agent or complete block of PLA2 enzyme by bromophenacyl bromide. These results indicate that the aggressive detaching effect of MLO venom is delivered not by an individual component of the venom, but rather is a combinatorial effect of several active ingredients.

Aim: To investigate the anti-inflammatory mechanism of the polysaccharides of Ginkgo biloba leaves (PGBL) by inhibiting leucocyte adhesion. Methods: The rough PGBL were isolated and purified. The anti-inflammatory effects of purified PGBL (p-PGBL) were assayed by ear edema induced by xylol and the acute peritonitis model in mice. The effect of p-PGBL on inhibiting the interaction between P-selectin and its ligands was investigated by flow cytometry and flow chamber. Results: p-PGBL could effectively inhibit the acute inflammation in mice and interfere with the adhesion of HL-60 cells, a human leukaemia cell line, or neutrophils to P-selectin in static conditions, as well as the adhesion of neutrophils to Chinese hamster ovary cells expressing human P-selectin and human umbilical vein endothelial cells in flow conditions in a dose-dependant manner. Conclusions: p-PGBL can inhibit the inflammatory process through interfering with the interaction between P-selectin and its ligands.

Blockage of the interaction of CD2 with its ligand CD58 is expected to bring out potential therapeutic value for autoimmune diseases and organ transplantation. Three series of peptides (cVL, cIL and cAQ series) were designed from ratCD2 and humanCD2 to modulate CD2–CD58 interaction. To determine the specific segments in parent peptides responsible for inhibitory activity as lead sequence, we generated shorter fragments of the parent peptides and evaluated their biological activity with cell adhesion assay. The structure–activity relationship studies indicated that small cyclic peptides derived from CD2 ligand binding epitopes could mimic native β-turn structure, and thus modulate CD2–CD58 interaction.

The effects of oxidative stress on integrin-mediated cell adhesion to the extracellular matrix (ECM) and related apoptosis were investigated using the EA.hy926 endothelial cells treated (or not) with two oxidants: the hypoxanthine/xanthine oxidase system (HX/XO) or the tert-butyl hydroperoxide (t-BHP) which both increased cell apoptosis. Cell adhesion onto vitronectin (Vn) and fibronectin (Fn) was increased at low concentrations of HX/XO (up to 5 mU/ml) or t-BHP (up to 125 microM) and prevented ROS-induced apoptosis. Flow cytometry analysis of integrin expression showed that the expression of integrin alphav and alpha5 subunits was, respectively, increased and decreased. Cell adhesion inhibition experiments using function-blocking monoclonal antibodies against integrin subunits indicated that alphavbeta1 and alphavbeta3 integrins were involved in adhesion of cells to Vn, and alphavbeta3 integrin played a major role in oxidant-treated cells. For adhesion to Fn, alpha5beta1 and alphavbeta1 integrins were required for oxidant-treated cells. Taken together, the results suggest that reactive oxygen species (ROS) produced either by HX/XO or t-BHP could affect expression and/or activation of specific integrins in the interaction of EA.hy926 cells with ECM.

In the mature gut, laminin-5 is expressed at the basal aspect of the differentiating epithelial cells. In vitro, we show that three more or less differentiated human colonic cancer HT29 cell lines produce and deposit laminin-5; they predominantly synthesize and secrete the 440 kDa form of laminin-5 that comprises the unprocessed 155 kDa gamma2 chain, as determined by immunoprecipitation analysis. In contrast, the highly differentiated colon carcinoma Caco-2 cells produce almost no laminin-5. Using anti-integrin antibodies, we show that adhesion of the two colonic cancer cell lines to laminin-5 is mediated by multiple integrin receptors including those for alpha3beta1, alpha6beta1 and alpha6beta4 integrins like in other cell types. In addition, the implication of integrin alpha2beta1 in this adhesion process is demonstrated for the first time. This has been shown by cell adhesion inhibition experiments, solid phase assays and confocal analysis. Together with previous in situ observations, these data provide a baseline knowledge for the understanding of the regulation of laminin-5 in normal and pathological intestine.In the mature gut, laminin-5 is expressed at the basal aspect of the differentiating epithelial cells. In vitro, we show that three more or less differentiated human colonic cancer HT29 cell lines produce and deposit laminin-5; they predominantly synthesize and secrete the 440 kDa form of laminin-5 that comprises the unprocessed 155 kDa gamma2 chain, as determined by immunoprecipitation analysis. In contrast, the highly differentiated colon carcinoma Caco-2 cells produce almost no laminin-5. Using anti-integrin antibodies, we show that adhesion of the two colonic cancer cell lines to laminin-5 is mediated by multiple integrin receptors including those for alpha3beta1, alpha6beta1 and alpha6beta4 integrins like in other cell types. In addition, the implication of integrin alpha2beta1 in this adhesion process is demonstrated for the first time. This has been shown by cell adhesion inhibition experiments, solid phase assays and confocal analysis. Together with previous in situ observations, these data provide a baseline knowledge for the understanding of the regulation of laminin-5 in normal and pathological intestine.

A contiguous four-guanosine (4G) sequence in c-myc antisense phosphorothioate oligonucleotides caused an antiproliferative effect in smooth muscle cells. To investigate the antiproliferative effect of c-myc antisense oligonucleotides on human lung cancer cell lines, we synthesized oligonucleotides of various lengths and sequences, focusing on the contiguous four-guanosine (4G) sequence. While a c-myc antisense oligonucleotide (20AS1 (4G)) targeted to the translation initiation codon of c-myc mRNA inhibited cell growth of A549 cells by 69% at 10 microM, a scrambled oligonucleotide (20SCR1 (4G)) containing the contiguous four-guanosine (4G) sequence also inhibited cell growth by 72% at the same dose. Although treatment with either 20AS1 (4G) or 20SCR1 (4G) inhibited cell adhesion by 70% at 10 microM, expression of c-myc protein was significantly suppressed only by 20AS1 (4G) (62%), and was only weakly inhibited by 20SCR1 (4G) (32%). Furthermore, a small cell lung carcinoma cell line, Lu65, which can grow in suspension form, was highly resistant to 20AS1 (4G) treatment (IC50>20 microM). These results suggest that the cell growth inhibition by c-myc antisense oligonucleotides containing the contiguous four-guanosine (4G) sequence was possibly correlated with inhibition of cell adhesion, but not with inhibition of c-myc protein expression, via a sequence-specific non-antisense mechanism.

Streptococcus suis, one of the most important and prevalent pathogens in swine, presents a major challenge to global public health. HP0197 is an S. suis surface antigen that was previously identified by immunoproteomics and can bind to the host cell surface. Here, we investigated the interaction between HP0197 and the host cell surface glycosaminoglycans (GAGs) using indirect immunofluorescence and cell adhesion inhibition assays. In addition, we determined that a novel 18-kDa domain in the N-terminal region of HP0197 functions as the GAG-binding domain. We then solved the three-dimensional structures of the N-terminal 18-kDa and C-terminal G5 domains using x-ray crystallography. Based on this structural information, the GAG-binding sites in HP0197 were predicted and subsequently verified using site-directed mutagenesis and indirect immunofluorescence. The results indicate that the positively charged residues on the exposed surface of the 18-kDa domain, which are primarily lysines, likely play a critical role in the HP0197-heparin interaction that mediates bacterium-host cell adhesion. Understanding this molecular mechanism may provide a basis for the development of effective drugs and therapeutic strategies for treating streptococcal infections. Background: The function of the Streptococcus suis antigen HP0197 is unknown, but it may be potentially involved in pathogenesis. Results: A novel GAG-binding domain on HP0197 was identified, and its structure was determined. Conclusion: Three clusters of basic residues on an 18-kDa domain of HP0197 are critical for heparin binding and cell adhesion. Significance: Understanding this mechanism will contribute to the design of drugs to treat streptococcal infections.

The architecture of the vascular wall is highly intricate and requires unique biomechanical properties in order to function properly. Native artery is composed of a mix of collagen, elastin, endothelial cells (ECs), smooth muscle cells (SMC), fibroblasts, and proteoglycans arranged into three distinct layers: the intima, media, and adventitia. Throughout artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery while undergoing pulsatile deformations [1]. The low-strain mechanical response of artery to blood flow is dominated by the elastic behavior of elastin which prevents pulsatile energy from being dissipated as heat [2]. Previous work has shown the ability to fabricate multi-layered electrospun scaffolds composed of polycaprolactone (PCL), elastin (ELAS), and collagen (COL), and their associated mechanical advantages. PCL was chosen, in this case, to provide mechanical integrity and elasticity, while elastin and collagen would provide further elasticity and bioactivity [3,4]. However, when the grafts were implanted in the descending aorta of a rat, cellular results were not as desirable as predicted. Therefore, further graft optimization was required. The hypothesis of this study was that blended polymers and biopolymers would be conducive for cellular attachment through specific integrin binding sites. To test this hypothesis, human umbilical artery smooth muscle cells (hUASMC) were seeded on electrospun PCL, COL, and ELAS blends for evaluation in a cell adhesion inhibition experiment.Copyright © 2011 by ASME

CD2 is a cell adhesion molecule that mediates T-cell activation by binding to its ligand CD58 on antigen-presenting cells. Interaction between CD2 and CD58 or leukocyte function-associated antigen-3 (LFA-3) helps to optimize immune recognition facilitating contact between T lymphocytes and antigen-presenting cells. Modulation or inhibition of this interaction has been shown to be therapeutically useful in the treatment of autoimmune diseases. Antibodies and small molecules including peptides have been designed to modulate or disrupt the cell adhesion interactions due to CD2 and CD58. E-rosetting assay is a widely used method applied in the study of the modulation of CD2-CD58 interaction, which is either labor-intensive or radio-hazardous. In this chapter, we describe two methods that are used to study cell adhesion inhibition: (a) E-rosetting Assay and (b) Lymphocyte-epithelial assay. The second method, lymphocyte-epithelial assay, is a rapid and sensitive heterotypic cell adhesion assay for studying cell adhesion inhibition. The method relies on the CD2 expression on the surface of Jurkat cells and the CD58 expression on the surface of Caco-2 cells, which were confirmed by flow cytometry and ELISA studies respectively. This heterotypic cell adhesion assay described typically takes less than 4 h to perform, allows the evaluation of inhibitory activity of peptides/small molecules to modulate CD2-CD58 interaction in real cell system.

Rat monoclonal antibodies were raised against fragment E3 of the mouse Engelbreth-Holm-Swarm (EHS) tumor laminin and selected according to their exclusive reaction with laminin A chain by immunoblotting and staining pattern in embryonic kidneys by immunofluorescence. Immunochemical studies of nine purified antibodies showed a comparable reaction with unfragmented laminin and fragment E3 but no cross-reaction with several other, unrelated laminin fragments including the major cell-binding fragment E8. Reduction or pepsin digestion of fragment E3 reduced or abolished antibody binding indicating that most of the epitopes involved are conformation dependent and do not include carbohydrates. They are, however, not identical as shown by different reactivities after proteolytic or chemical cleavage of E3. Four of the antibodies were highly active in inhibiting cell adhesion of the teratocarcinoma cell line F9 and the Schwannoma cell line RN22 on fragment E3 (IC50 approximately 1 microgram/ml), while the others were distinctly less active. No inhibition was observed for cell adhesion on unfragmented laminin, consistent with previous findings that this is largely mediated by binding of fragment E8 to alpha 6 beta 1 integrin. A distinct correlation was observed between cell adhesion inhibition and the inhibition of heparansulfate proteoglycan and heparin binding to fragment E3. Since heparin is not very efficient in inhibiting cell adhesion, it indicates that heparin- and cell-binding sites on fragment E3 are in close proximity but not identical. Two of the antibodies also showed partial inhibition of kidney tubule formation in organ culture of embryonic kidney mesenchyme while the other antibodies were inactive. It suggests some but probably minor involvement of the fragment E3 structure of laminin in this developmental process.

Eight alkamides 1-8 were isolated by bioassay-guided isolation of EtOH extracts of the fruits of Piper longum and Piper nigum (Piperaceae). Their structures were elucidated by spectroscopic analysis ((1)H, (13)C NMR, and ESI-MS) as follows: guineensine (1), retrofracamide C (2), (2E,4Z,8E)-N-[9-(3,4-methylenedioxyphenyl)-2,4,8-nonatrienoyl]piperidine (3), pipernonaline (4), piperrolein B (5), piperchabamide D (6), pellitorin (7), and dehydropipernonaline (8). Their compounds 3-5, 7, and 8 inhibited potently the direct binding between sICAM-1 and LFA-1 of THP-1 cells in a dose-dependent manner, with IC(50) values of 10.7, 8.8, 13.4, 13.5, and 6.0 microg/mL, respectively.

The Thomsen-Friedenreich antigen (TF) is a pancarcinoma marker which is involved in the development of liver metastasis by binding tumour cells to the asialoglycoprotein receptor on hepatocytes. Blocking of this receptor prevents metastasis under certain circumstances. We report on conditions for an effective inhibition of the adhesion of KG-1 leukaemia cells expressing TF by lactosylated liposomes. In order to reach strong inhibition, carbohydrate blocking probes must be multivalent. Glycoliposomes are able to carry a large number of glycolipids accommodated in the lipid bilayer. They should be able to adapt their glycolipid pattern in order to achieve multiple binding. We found that, in addition to the number of carbohydrates on the liposome surface, their size, and probably the arrangement of neutral glycolipids in clustered domains, determine the inhibitory properties of glycoliposomes.

Objective: Hydroxiapatite (HA) has been suggested as a useful biomaterial to support the regeneration of tissues. In this study, we investigated the adhesion of periodontal ligament (PDL) cells on octacalcium phosphate (OCP) and its hydrolyzed apatitic product (HL), which are known precursors of HA. Methods: Rat PDL cells were cultured on OCP or HL-coated dishes. Cell proliferation and adhesion and mRNA expression of collagen I, fibronectin integrin subunits were examined. Cell adhesion inhibition assays were carried out by GRGDSPK (Gly-Arg-Gly-Asp-Ser-Pro-Lys). Results: In early culture period, the cell number of PDL cells was lower on OCP and HL than that on control without any coating. However, the cell number on OCP or HL caught up with control later period. mRNA expression level of collagen I and fibronectin on OCP and HL were similar among OCP HL and control, although they differed early in the culture period. Integrin subunits were expressed on both OCP and HL as well as on control. Cell adhesion was inhibited by RGD inhibitor peptide. Conclusion: Our findings indicated that rat PDL cells produce collagen I and fibronectin on OCP and HL, and then show increased cell numbers depending on adhesion to the matrices through integrins.

Ionizing radiation (IR) therapy is one of the main therapies to treat breast cancer, but accumulating evidence suggests that IR is potentially associated with increased invasion and metastasis. A great increase in breast cancer cell adhesion to fibronectin after high dose α-radiation was observed in our group, and it accompanied with ROS boost. However it is not known whether ROS increase related to the cell adhesion increase after α-radiation. In this report, we showed that the increased cell adhesion to fibronectin was reduced by ROS scavenger L-NAC by 21.8% after α-radiation, but was not affected by H2O2 scavenger, catalase or by cNOS inhibitor, L-NAME with 24 h treatment. We then determined that the cell adhesion inhibition after α-radiation involve reduced ROS, and reduced vinculin expression and integrin activation after treating with L-NAC for 24 h. However, a short-term, 1 h pre-IR followed by 2 h post-IR, treatment of L-NAC promotes ROS production and has no effect on cell adhesion at 24 h post-IR. These results suggest that L-NAC long-term treatment could potentially have an impact on breast cancer metastasis by inhibiting the ROS and cell adhesion. These results suggest that to inhibit IR-induced breast cancer adhesion, an uninterrupted treatment of L-NAC may be required. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4342. doi:1538-7445.AM2012-4342

International audience; Acid-soluble and pepsin-treated collagen V were prepared from fetal human bones or human placenta, respectively, to be tested for potential cell adhesion promoting activity. Out of 14 different collagen I-adhering cell lines, 10 showed distinct adhesion to collagen V. In all cases adhesion was followed by spreading. The activities of intact and pepsin-solubilized collagen V were similar, suggesting that the cell binding sites are restricted to the triple-helical domain of the molecules. Cell adhesion was also induced by the unfolded form of collagen V and after separation of the alpha chains by heparin affinity chromatography. Isolated alpha 2(V) chains, rich in RGD sequences, were more efficient than isolated alpha 1(V) chains. However, cell adhesion to native or denatured collagen V did not proceed by the same molecular mechanisms as shown by cell adhesion inhibition experiments. Cell adhesion to native collagen V was insensitive to the presence of RGD-containing synthetic peptides while adhesion to denatured collagen V was inhibited by the peptides. Furthermore, the results strongly suggested a major role for alpha 1 beta 1 and alpha 2 beta 1 integrins in the RGD-independent cell adhesion to native collagen V. These data indicate that collagen V is a specific adhesive substrate for different cell types. It also suggests that distinct sets of RGD-dependent and RGD-independent receptors mediate cell attachment to unfolded and native collagen V, respectively. This mechanism is shared by at least the interstitial collagens I and VI, which supports the hypothesis that when included in the triple-helical conformation of collagens, RGD sequences are either not accessible to cells or exhibit specific conformations recognized by different integrins.Acid-soluble and pepsin-treated collagen V were prepared from fetal human bones or human placenta, respectively, to be tested for potential cell adhesion promoting activity. Out of 14 different collagen I-adhering cell lines, 10 showed distinct adhesion to collagen V. In all cases adhesion was followed by spreading. The activities of intact and pepsin-solubilized collagen V were similar, suggesting that the cell binding sites are restricted to the triple-helical domain of the molecules. Cell adhesion was also induced by the unfolded form of collagen V and after separation of the alpha chains by heparin affinity chromatography. Isolated alpha 2(V) chains, rich in RGD sequences, were more efficient than isolated alpha 1(V) chains. However, cell adhesion to native or denatured collagen V did not proceed by the same molecular mechanisms as shown by cell adhesion inhibition experiments. Cell adhesion to native collagen V was insensitive to the presence of RGD-containing synthetic peptides while adhesion to denatured collagen V was inhibited by the peptides. Furthermore, the results strongly suggested a major role for alpha 1 beta 1 and alpha 2 beta 1 integrins in the RGD-independent cell adhesion to native collagen V. These data indicate that collagen V is a specific adhesive substrate for different cell types. It also suggests that distinct sets of RGD-dependent and RGD-independent receptors mediate cell attachment to unfolded and native collagen V, respectively. This mechanism is shared by at least the interstitial collagens I and VI, which supports the hypothesis that when included in the triple-helical conformation of collagens, RGD sequences are either not accessible to cells or exhibit specific conformations recognized by different integrins.

We have investigated the structure-activity relationship of a series of new synthetic RGD analogs and their potential use as specific platelet aggregation inhibitors. Twelve short linear peptides showed high potency to inhibit aggregation in ADP-stimulated dog platelets. In order to assess the selectivity of these analogs towards platelet integrin GPIIb-IIIa, a new cell adhesion inhibition system was devised which was able to discriminate between the two closely related beta 3-integrins of the vasculature, GPIIb-IIIa (alpha IIb beta 3), present in platelets, and the vitronectin receptor (alpha v beta 3), expressed in endothelial cells and platelets. As reported for other peptides by Scarborough et al. (1993, J. Biol. Chem. 268, 1066), the analogs containing lysine instead of arginine in position 1 showed increased selectivity towards GPIIb-IIIa. One of them, in which the piperidine carboxylic group was attached to the N-terminus of KGDW, not only strongly inhibited platelet aggregation, but also selectively abolished cell adhesion mediated by GPIIb-IIIa without effect on the vitronectin receptor.