Interactions via α2ß1 Cell Integrin May Protect against the Progression of Airway Structural Changes in Asthma.

Increased airway wall thickness and remodeling of bronchial mucosa are characteristic of asthma and may arise from altered integrin signaling on airway cells. Here, we analyzed the expression of ß1-subfamily integrins on blood and airway cells (flow cytometry), inflammatory biomarkers in serum and bronchoalveolar lavage, reticular basement membrane (RBM) thickness and collagen deposits in the mucosa (histology), and airway geometry (CT-imaging) in 92 asthma patients (persistent airflow limitation subtype: n = 47) and 36 controls. Persistent airflow limitation was associated with type-2 inflammation, elevated soluble α2 integrin chain, and changes in the bronchial wall geometry. Both subtypes of asthma showed thicker RBM than control, but collagen deposition and epithelial α1 and α2 integrins staining were similar. Type-I collagen accumulation and RBM thickness were inversely related to the epithelial expression of the α2 integrin chain. Expression of α2ß1 integrin on T-cells and eosinophils was not altered in asthma. Collagen I deposits were, however, more abundant in patients with lower α2ß1 integrin on blood and airway CD8+ T-cells. Thicker airway walls in CT were associated with lower α2 integrin chain on blood CD4+ T-cells and airway eosinophils. Our data suggest that α2ß1 integrin on inflammatory and epithelial cells may protect against airway remodeling advancement in asthma.

Lebein, a snake venom disintegrin, suppresses human colon cancer cells proliferation and tumor-induced angiogenesis through cell cycle arrest, apoptosis induction and inhibition of VEGF expression.

Lebein, is an heterodimeric disintegrin isolated from Macrovipera lebetina snake venom that was previously characterized as an inhibitor of ADP-induced platelet aggregation. In this study, we investigated the effect of Lebein on the p53-dependent growth of human colon adenocarcinoma cell lines. We found that Lebein significantly inhibited LS174 (p53wt), HCT116 (p53wt), and HT29 (p53mut) colon cancer cell viability by inducing cell cycle arrest through the modulation of expression levels of the tumor suppression factor p53, cell cycle regulating proteins cyclin D1, CDK2, CDK4, retinoblastoma (Rb), CDK1, and cyclin-dependent kinase inhibitors p21 and p27. Interestingly, Lebein-induced apoptosis of colon cancer cells was dependent on their p53 status. Thus, in LS174 cells, cell death was associated with PARP cleavage and the activation of caspases 3 and 8 while in HCT116 cells, Lebein induced caspase-independent apoptosis through increased expression of apoptosis inducing factor (AIF). In LS174 cells, Lebein triggers the activation of the MAPK ERK1/2 pathway through induction of reactive oxygen species (ROS). It also decreased cell adhesion and migration to fibronectin through down regulation of α5ß1 integrin. Moreover, Lebein significantly reduced the expression of two angiogenesis stimulators, Vascular Endothelial Growth Factor (VEGF) and Neuropilin 1 (NRP1). It inhibited the VEGF-induced neovascularization process in the quail embryonic CAM system and blocked the development of human colon adenocarcinoma in nude mice. Overall, our work indicates that Lebein may be useful to design a new therapy against colon cancer. © 2016 Wiley Periodicals, Inc.

Soft Substrates Containing Hyaluronan Mimic the Effects of Increased Stiffness on Morphology, Motility, and Proliferation of Glioma Cells.

Unlike many other cancer cells that grow in tumors characterized by an abnormally stiff collagen-enriched stroma, glioma cells proliferate and migrate in the much softer environment of the brain, which generally lacks the filamentous protein matrix characteristic of breast, liver, colorectal, and other types of cancer. Glial cell-derived tumors and the cells derived from them are highly heterogeneous and variable in their mechanical properties, their response to treatments, and their properties in vitro. Some glioma samples are stiffer than normal brain when measured ex vivo, but even those that are soft in vitro stiffen after deformation by pressure gradients that arise in the tumor environment in vivo. Such mechanical differences can strongly alter the phenotype of cultured glioma cells. Alternatively, chemical signaling might elicit the same phenotype as increased stiffness by activating intracellular messengers common to both initial stimuli. In this study the responses of three different human glioma cell lines to changes in substrate stiffness are compared with their responses on very soft substrates composed of a combination of hyaluronic acid and a specific integrin ligand, either laminin or collagen I. By quantifying cell morphology, stiffness, motility, proliferation, and secretion of the cytokine IL-8, glioma cell responses to increased stiffness are shown to be nearly identically elicited by substrates containing hyaluronic acid, even in the absence of increased stiffness. PI3-kinase activity was required for the response to hyaluronan but not to stiffness. This outcome suggests that hyaluronic acid can trigger the same cellular response, as can be obtained by mechanical force transduced from a stiff environment, and demonstrates that chemical and mechanical features of the tumor microenvironment can achieve equivalent reactions in cancer cells.

Anti-angiogenic activities of snake venom CRISP isolated from Echis carinatus sochureki.

BACKGROUND: Cysteine-rich secretory protein (CRISP) is present in majority of vertebrate including human. The physiological role of this protein is not characterized. We report that a CRISP isolated from Echis carinatus sochureki venom (ES-CRISP) inhibits angiogenesis. METHODS: The anti-angiogenic activity of purified ES-CRISP from snake venom was investigated in vitro using endothelial cells assays such as proliferation, migration and tube formation in Matrigel, as well as in vivo in quail embryonic CAM system. The modulatory effect of ES-CRISP on the expression of major angiogenesis factors and activation of angiogenesis pathways was tested by qRT-PCR and Western blot. RESULTS: The amino acid sequence of ES-CRISP was found highly similar to other members of this snake venom protein family, and shares over 50% identity with human CRISP-3. ES-CRISP supported adhesion to endothelial cells, although it was also internalized into the cytoplasm in a granule-like manner. It blocked EC proliferation, migration and tube formation in Matrigel. In the embryonic quail CAM system, ES-CRISP abolished neovascularization process induced by exogenous growth factors (bFGF, vpVEGF) and by developing gliomas. CRISP modulates the expression of several factors at the mRNA level, which were characterized as regulators of angiogenesis and blocked activation of MAPK Erk1/2 induced by VEGF. CONCLUSIONS: ES-CRISP was characterized as a negative regulator of the angiogenesis, by direct interaction with endothelial cells. GENERAL SIGNIFICANCE: The presented work may lead to the development of novel angiostatic therapy, as well as contribute to the identification of the physiological relevance of this functionally uncharacterized protein.

Vimocin and vidapin, cyclic KTS peptides, are dual antagonists of α1ß1/α2ß1 integrins with antiangiogenic activity.

Obtustatin and viperistatin, members of the disintegrin protein family, served as lead compounds for the synthesis of linear and cyclic peptides containing the KTS binding motif. The most active linear peptide, a viperistatin analog, indicated the importance of Cys(19) and Cys(29), as well as the presence of Arg at position 24 for their biologic activity, and was used as the basic sequence for the synthesis of cyclic peptides. Vimocin (compound 6) and vidapin (compound 10) showed a high potency (IC50 = 0.17 nM) and intermediate efficacy (20 and 40%) in inhibition of adhesion of α1/α2 integrin overexpressor cells to respective collagens. Vimocin was more active in inhibition of the wound healing (53%) and corneal micropocket (17%) vascularization, whereas vidapin was more potent in inhibition of migration in the Matrigel tube formation assay (90%). Both compounds similarly inhibited proliferation (50-90%) of endothelial cells, and angiogenesis induced by vascular endothelial growth factor (80%) and glioma (55%) in the chorioallantoic membrane assay. These peptides were not toxic to endothelial cell cultures and caused no acute toxicity upon intravenous injection in mice, and were stable for 10-30 hours in human serum. The in vitro and in vivo potency of the peptides are consistent with conformational ensembles and "bioactive" space shared by obtustatin and viperistatin. These findings suggest that vimocin and vidapin can serve as dual α1ß1/α2ß1 integrin antagonists in antiangiogenesis and cancer therapy.

Compression stiffening of brain and its effect on mechanosensing by glioma cells.

Many cell types, including neurons, astrocytes and other cells of the central nervous system respond to changes in extracellular matrix or substrate viscoelasticity, and increased tissue stiffness is a hallmark of several disease states including fibrosis and some types of cancers. Whether the malignant tissue in brain, an organ that lacks the protein-based filamentous extracellular matrix of other organs, exhibits the same macroscopic stiffening characteristic of breast, colon, pancreatic, and other tumors is not known. In this study we show that glioma cells like normal astrocytes, respond strongly in vitro to substrate stiffness in the range of 100 to 2000 Pa, but that macroscopic (mm to cm) tissue samples isolated from human glioma tumors have elastic moduli on the order of 200 Pa that are indistinguishable from those of normal brain. However, both normal brain and glioma tissues increase their shear elastic moduli under modest uniaxial compression, and glioma tissue stiffens more strongly under compression than does normal brain. These findings suggest that local tissue stiffness has the potential to alter glial cell function, and that stiffness changes in brain tumors might arise not from increased deposition or crosslinking of collagen-rich extracellular matrix but from pressure gradients that form within the tumors in vivo.

Blocking of α1ß1 and α2ß1 adhesion molecules inhibits eosinophil migration through human lung microvascular endothelial cell monolayer.

INTRODUCTION: In cell trafficking to the airways in asthma, among integrins the most important are those containing α4 and ß2 subunits. We have previously shown that also blocking of collagen receptors, α1ß1 and α2ß1 integrins, inhibits transmigration of eosinophils of asthmatic subjects through a monolayer of skin microvascular endothelial cells seeded on collagen IV coated inserts. However, it was not clear whether this observation was limited to asthma or depended on the type of microvascular cell and collagen IV used as a base. MATERIALS & METHODS: In the current study we performed a transmigration assay using human lung microvascular endothelial cells seeded directly on a plastic surface as a base and blood cells isolated from 12 representatives of each of two groups, asthmatics and healthy donors, by gradient centrifugation, followed by immunomagnetic negative separation of eosinophils. Isolated eosinophils and peripheral blood mononuclear cells (PBMC) were inhibited by snake venom-derived integrin antagonists including viperistatin and VP12, as inhibitors of α1ß1 and α2ß1 integrin, respectively, and VLO5 and VLO4, as inhibitors of α4ß1 and α5ß1 integrin, respectively. RESULTS: All snake venom-derived anti-adhesive proteins were effective in inhibiting eosinophil transmigration, whilst only VLO5 and VLO4 reduced PBMC mobility in this assay. This observation was similar in both groups of subjects studied. DISCUSSION: α1ß1 and α2ß1 integrins could be involved in transmigration of eosinophil to the inflammatory site. Migratory inhibition was observed in asthma subjects as well as in healthy donors, and did not depend on origin of endothelial cells or the extracellular matrix component used as a base.

Structure-Activity Relationship of Synthetic Linear KTS-Peptides Containing Meta-Aminobenzoic Acid as Antagonists of α1ß1 Integrin with Anti-Angiogenic and Melanoma Anti-Tumor Activities.

To develop peptide drugs targeting integrin receptors, synthetic peptide ligands endowed with well-defined selective binding motifs are necessary. The snake venom KTS-containing disintegrins, which selectively block collagen α1ß1 integrin, were used as lead compounds for the synthesis and structure-activity relationship of a series of linear peptides containing the KTS-pharmacophore and alternating natural amino acids and 3-aminobenzoic acid (MABA). To ensure a better stiffness and metabolic stability, one, two and three MABA residues, were introduced around the KTS pharmacophore motif. Molecular dynamics simulations determined that the solution conformation of MABA peptide 4 is more compact, underwent larger conformational changes until convergence, and spent most of the time in a single cluster. The peptides' binding affinity has been characterized by an enzyme linked immunosorbent assay in which the most potent peptide 4 inhibited with IC50 of 324 ± 8 µM and 550 ± 45 µM the binding of GST-α1-A domain to collagen IV fragment CB3, and the cell adhesion to collagen IV using α1-overexpressor cells, respectively. Docking studies and MM-GBSA calculations confirmed that peptide 4 binds a smaller region of the integrin near the collagen-binding site and penetrated deeper into the binding site near Trp1. Peptide 4 inhibited tube formation by endothelial cell migration in the Matrigel angiogenesis in vitro assay. Peptide 4 was acutely tolerated by mice, showed stability in human serum, decreased tumor volume and angiogenesis, and significantly increased the survival of mice injected with B16 melanoma cells. These findings propose that MABA-peptide 4 can further serve as an α1ß1-integrin antagonist lead compound for further drug optimization in angiogenesis and cancer therapy.

Fibronectin extra domain-A promotes hepatic stellate cell motility but not differentiation into myofibroblasts.

BACKGROUND & AIMS: Myofibroblasts are the primary cell type involved in physiologic wound healing and its pathologic counterpart, fibrosis. Cellular fibronectin that contains the alternatively spliced extra domain A (EIIIA) is up-regulated during these processes and is believed to promote myofibroblast differentiation. We sought to determine the requirement for EIIIA in fibrosis and differentiation of myofibroblasts in rodent livers. METHODS: We used a mechanically tunable hydrogel cell culture system to study differentiation of primary hepatic stellate cells and portal fibroblasts from rats into myofibroblasts. Liver fibrosis was induced in mice by bile duct ligation or administration of thioacetamide. RESULTS: EIIIA was not required for differentiation of rat hepatic stellate cells or portal fibroblasts into fibrogenic myofibroblasts. Instead, hepatic stellate cells cultured on EIIIA-containing cellular fibronectin formed increased numbers of lamellipodia; their random motility and chemotaxis also increased. These increases required the receptor for EIIIA, the integrin α(9)ß(1). In contrast, the motility of portal fibroblasts did not increase on EIIIA, and these cells expressed little α(9)ß(1). Male EIIIA(-/-) mice were modestly protected from thioacetamide-induced fibrosis, which requires motile hepatic stellate cells, but not from bile duct ligation-induced fibrosis, in which portal fibroblasts are more important. Notably, myofibroblasts developed during induction of fibrosis with either thioacetamide or bile duct ligation in EIIIA(-/-) mice. CONCLUSIONS: EIIIA is dispensable for differentiation of hepatic stellate cells and portal fibroblasts to myofibroblasts, both in culture and in mouse models of fibrosis. Our findings, however, indicate a role for EIIIA in promoting stellate cell motility and parenchymal liver fibrosis.

Identification of inhibitors of α2ß1 integrin, members of C-lectin type proteins, in Echis sochureki venom.

Snake venom antagonists of α2ß1 integrin have been identified as members of a C-lectin type family of proteins (CLP). In the present study, we characterized three new CLPs isolated from Echis sochureki venom, which interact with this integrin. These proteins were purified using a combination of gel filtration, ion exchange chromatography and reverse phase HPLC. Sochicetin-A and sochicetin-B potently inhibited adhesion of cells expressing α2ß1 integrin and binding of isolated α2ß1 ectodomain to collagen I, as well as bound to recombinant GST-α2A domain in ELISA, whereas activity of sochicetin-C in these assays was approximately two orders of magnitude lower. Structurally, sochicetin-B and sochicetin-C are typical heterodimeric αß CLPs, whereas sochicetin-A exhibits a trimer of its subunits (αß)3 in the quaternary structure. Immobilized sochicetins supported adhesion of glioma cell lines, LN18 and LBC3, whereas in a soluble form they partially inhibited adhesion of these cells to collagen I. Glioma cells spread very poorly on sochicetin-A, showing no cytoskeleton rearrangement typical for adhesion to collagen I or fibronectin. Adhesion on CLP does not involve focal adhesion elements, such as vinculin. Sochicetin-A also inhibited collagen-induced platelet aggregation, similar to other CLPs' action on the blood coagulation system.

NGF promotes hemodynamic recovery in a rabbit hindlimb ischemic model through trkA- and VEGFR2-dependent pathways.

Nerve growth factor (NGF) has been reported to play an important role in physiological and pathological angiogenesis. Based on these observations, we hypothesized that NGF may induce the formation of functional blood vessels in a hindlimb ischemic rabbit model. Hindlimb ischemia was induced in 34 rabbits bilaterally by endovascular embolization of femoral arteries. On the 7th, 14th, and 20th postembolization days, NGF was injected intramuscularly, in 1 ischemic limb, and vehicle was injected in the contralateral control limb. On the 40th day, newly developed collateral vessels (diameter >500 µm) were quantified by transauricular intraarterial subtraction angiography. Perfusion analysis of an in vivo dynamic computed tomography study was performed to the limbs to investigate the hemodynamic recovery of the distal ischemic tissues. Functional estimation of limb perfusion showed a statistically significant increase of blood flow and blood volume for NGF. However, the increase of the collateral vessels was not detectable angiographically, providing evidence for the existence of a NGF-stimulated capillary angiogenic network but not increase of arteriogenesis. The combination of NGF with either tropomyosin-related kinase type A or vascular endothelial growth factor receptor 2 antagonists abolished the NGF-induced hemodynamic recovery. These findings provide new insights into understanding the involvement of NGF in vascular formation and its applications in therapeutic angiogenesis.

Effect of Dimeric Disintegrins Isolated from Vipera lebetina obtusa Venom on Glioblastoma Cellular Responses.

Integrins play a fundamental role in the migration and invasiveness of glioblastoma (GBM) cells, making them suitable targets for innovative cancer therapy. The aim of this study was to evaluate the effect of the RGD homodimeric disintegrin VLO4, isolated from Vipera lebetina obtusa venom, on the adhesion, spreading, migration, and survival of LBC3, LN18, and LN229 cell lines. This disintegrin, as a potent antagonist for α5ß1 integrin, showed pro-adhesive properties for these cell lines, the highest for LN229 and the lowest for LBC3. Glioblastoma cells displayed significant differences in the spreading on the immobilized VLO4 and the natural α5ß1 integrin ligand, fibronectin. Solubilized VLO4 showed different cytotoxicity and pro-apoptotic properties among tested cell lines, with the highest against LN18 and none against LN229. Moreover, VLO4 revealed an inhibitory effect on the migration of LBC3 and LN18 cell lines, in contrast to LN229 cells, which were not sensitive to this disintegrin. However, LN229 migration was impaired by VLO5, a disintegrin antagonistic to integrin α9ß1, used in combination with VLO4. A possible mechanism of action of VLO4 may be related to the downregulation of α5ß1 integrin subunit expression, as revealed by Western blot. VLO4 also inhibited cell proliferation and induced caspase-dependent apoptosis in LBC3 and LN18 cell lines. These results indicate that targeting α5ß1 integrin by related VLO4 compounds may be useful in the development of integrin-targeted therapy for glioblastoma.

alpha(9)beta(1) integrin engagement inhibits neutrophil spontaneous apoptosis: involvement of Bcl-2 family members.

Integrin signaling is comprised of well-characterized pathways generally involved in cell survival. alpha(9)beta(1) integrin has recently become a target of study and has been shown to present pro-survival effects on neutrophils. However, there are no detailed studies on how alpha(9)beta(1) integrin-coupled signaling pathways interact and how they converge to finally modulate spontaneous apoptosis in neutrophils. In this regard we sought to investigate the main signaling events triggered by alpha(9)beta(1) integrin engagement and how these signaling pathways modulate the apoptotic program of human neutrophils. Using VLO5, a snake venom disintegrin shown to bind to alpha(9)beta(1) integrin in neutrophils, we demonstrate that alpha(9)beta(1) integrin engagement leads to the activation of integrin signaling pathways and potently reduces neutrophil spontaneous apoptosis. These effects are dependent on the activation of PI3K and MAPK pathways, since both LY294002 (PI3K inhibitor) or PD95059 (MEK inhibitor) reverted the effects of VLO5/alpha(9)beta(1) interaction. Moreover we show that VLO5/alpha(9)beta(1) engagement induces NF-kappaB nuclear translocation and increases the ratio between anti- and pro-apoptotic proteins by inducing the degradation of pro-apoptotic protein Bad and increasing the expression of anti-apoptotic protein Bcl-x(L). VLO5 also inhibited the early steps of neutrophil spontaneous apoptosis by preventing Bax translocation to the outer mitochondrial membrane and consequent cytochrome c release. In conclusion, as the mechanistic details of alpha(9)beta(1) integrin signaling pathways in human neutrophils becomes clearer, it should become possible to develop new therapeutic agents for human diseases where neutrophils play a prominent role.

NMR structure and dynamics of recombinant wild type and mutated jerdostatin, a selective inhibitor of integrin α1ß1.

NMR analysis of four recombinant jerdostatin molecules was assessed to define the structural basis of two naturally occurring gain-of-function events: C-terminal dipeptide processing and mutation of the active residue K21 to arginine. Removal of the highly mobile and a bulky C-terminal dipeptide produced pronounced chemical shift changes in the sequentially unconnected but spatially nearby α(1)ß(1) inhibitory loop. Analysis of chemical shift divergence and (15)N backbone relaxation dynamics indicated differences in motions in the picosecond to nanosecond time scale, and the higher T(2) rate of S25, S26, and H27 of rJerK21 point to a slowdown in the microsecond to millisecond motions of these residues when compared with rJerR21. The evidence presented in this article converges on the hypothesis that dynamic differences between the α(1)ß(1) recognition loops of rJerR21 and rJerK21 may influence the thermodynamics of their receptor recognition and binding. A decrease in the µs-ms time scale may impair the binding affinity by reducing the rate of possible conformations that the rJerK21 can adopt in this time scale.

Neurotrophic factors and their receptors in lung development and implications in lung diseases.

Although lung innervation has been described by many studies in humans and rodents, the regulation of the respiratory system induced by neurotrophins is not fully understood. Here, we review current knowledge on the role of neurotrophins and the expression and function of their receptors in neurogenesis, vasculogenesis and during the embryonic development of the respiratory tree and highlight key implications relevant to respiratory diseases.

Synthesis and Pharmacological Characterization of Visabron, a Backbone Cyclic Peptide Dual Antagonist of α4ß1 (VLA-4)/α9ß1 Integrin for Therapy of Multiple Sclerosis.

Integrins α4ß1/ α9ß1 are important in the pathogenesis and progression of inflammatory and autoimmune diseases by their roles in leukocyte activation and trafficking. Natalizumab, a monoclonal antibody selectively targeting α4ß1 integrin and blocking leukocyte trafficking to the central nervous system, is an immunotherapy for multiple sclerosis (MS). However, due to its adverse effects associated with chronic treatment, alternative strategies using small peptide mimetic inhibitors are being sought. In the present study, we synthesized and characterized visabron c (4-4), a backbone cyclic octapeptide based on the sequence TMLD, a non-RGD unique α4ß1 integrin recognition sequence motif derived from visabres, a proteinous disintegrin from the viper venom. Visabron c (4-4) was selected from a minilibrary with conformational diversity based on its potency and selectivity in functional adhesion cellular assays. Visabron c (4-4)'s serum stability, pharmacokinetics, and therapeutic effects following ip injection were assessed in an experimental autoimmune encephalomyelitis (EAE) animal model. Furthermore, visabron c (4-4)'s lack of toxic effects in mice was verified by blood analysis, tissue pathology, immunogenicity, and "off-target" effects, indicating its significant tolerability and lack of immunogenicity. Visabron c (4-4) can be delivered systemically. The in vitro and in vivo data justify visabron c (4-4) as a safe alternative peptidomimetic lead compound/drug to monoclonal anti-α4 integrin antibodies, steroids, and other immunosuppressant drugs. Moreover, visabron c (4-4) design may pave the way for developing new therapies for a variety of other inflammatory and/or autoimmune diseases.

Pharmacodynamic Studies of Fluorescent Diamond Carriers of Doxorubicin in Liver Cancer Cells and Colorectal Cancer Organoids.

BACKGROUND: We recently reported on preferential deposition of bare fluorescent diamond particles FDP-NV-700/800nm (FDP-NV) in the liver following intravenous administration to rats. The pharmacokinetics of FDP-NV in that species indicated short residency in the circulation by rapid clearance by the liver. Retention of FDP-NV in the liver was not associated with any pathology. These observations suggested that cancer therapeutics, such as doxorubicin, linked to FDP-NV, could potentially serve for anti-cancer treatment while sparing toxicities of peripheral organs. PURPOSE: To generate proof-of-concept (POC) and detail mechanisms of action of doxorubicin-coated FDP-NV-700/800nm (FDP-DOX) as a prospective chemotherapeutic for metastatic liver cancer. METHODS: FDP-DOX was generated by adsorption chemistry. Experimental design included concentration and time-dependent efficacy studies as compared with naïve (baren) FDP-NV in in vitro liver cancer cells models. Uptake of FDP-NV and FDP-DOX by HepG-2, Hep-3B and hCRC organoids were demonstrated by flow-cytometry and fluorescent microscopy. FDP-DOX pharmacodynamic effects included metabolic as well as cell death biomarkers Annexin V, TUNEL and LDH leakage. DOX desorpted from FDP-DOX was assessed by confocal microscopy and chemical assay of cells fractions. RESULTS: FDP-DOX efficacy was dose- and time-dependent and manifested in both liver cancer cell lines and human CRC organoids. FDP-DOX was rapidly internalized into cancer cells/organoids leading to cancer growth inhibition and apoptosis. FDP-DOX disrupted cell membrane integrity as evident by LDH release and suppressing mitochondrial metabolic pathways (AlamarBlue assay). Access of free DOX to the nuclei was confirmed by direct UV-Visible fluorescent assay and confocal microscopy of DOX fluorescence. CONCLUSION: The rapid uptake and profound cancer inhibition observed using FDP-DOX in clinically relevant cancer models, highlight FDP-DOX promise for cancer chemotherapeutics. We also conclude that the in vitro data justify further investment in in vivo POC studies.

Structural requirements of KTS-disintegrins for inhibition of alpha(1)beta(1) integrin.

Obtustatin and viperistatin represent the shortest known snake venom monomeric disintegrins. In the present study, we have produced recombinant full-length wild-type and site-directed mutants of obtustatin to assess the role of the K(21)TS(23) tripeptide and C-terminal residues for specific inhibition of the alpha(1)beta(1) integrin. Thr(22) appeared to be the most critical residue for disintegrin activity, whereas substitution of the flanking lysine or serine residues for alanine resulted in a less pronounced decrease in the anti-alpha(1)beta(1) integrin activity of the disintegrin. The triple mutant A(21)AA(23) was devoid of blocking activity towards alpha(1)beta(1) integrin-mediated cell adhesion. The potency of recombinant KTS-disintegrins also depended on the residue C-terminally adjacent to the active motif. Substitution of Leu(24) of wild-type obtustatin for an alanine residue slightly decreased the inhibitory activity of the mutant, whereas an arginine residue in this position enhanced the potency of the mutant over wild-type obtustatin by 6-fold. In addition, the replacements L38V and P40Q may account for a further 25-fold increase in alpha(1)beta(1) inhibitory potency of viperistatin over KTSR-obtustatin.

Cell-Based Adhesion Assays for Isolation of Snake Venom's Integrin Antagonists.

Snake venoms could lead to the development of new drugs to treat a range of life-threatening conditions like cardiovascular diseases. Most snake venoms contain a large variety of lethal toxins as well as anti-adhesive proteins such as disintegrins, which have evolved from the harmless compounds ADAMs (proteins with a disintegrin and a metalloprotease domain) and C-type lectin proteins which disturb connective tissue and cell-matrix interaction. These anti-adhesive proteins target and block integrin receptors and disrupt normal biological processes in snakes' prey such as connective tissue physiology and blood clotting. This chapter provides the experimental details of a practical, cell-based adhesion protocol to help identify and isolate disintegrins and C-type lectin proteins from snake venoms, important tools in integrin research and lead compounds for drug discovery.

Effects of Fluorescent Diamond Particles FDP-NV-800nm on Essential Biochemical Functions of Primary Human Umbilical Vein Cells and Human Hepatic Cell Line, HepG-2 in vitro (Part VI): Acute Biocompatibility Studies.

BACKGROUND: Recently, we reported the safety and biocompatibility of fluorescent diamond particles, FDP-NV-Z-800nm (FDP-NV) injected intravenously into rats, where no morbidity and mortality were noted over a period of 3 months. The acute effects of FDP-NV-800nm particles on cultured human endothelial and hepatic cells remain unexplored. PURPOSE: In this study, we aimed to explore select cellular and biochemical functions in cultured human umbilical endothelial cells (HUVEC) and a human hepatic cancer cell line (HepG-2) exposed to FDP-NV-800 in vitro at exposure levels within the pharmacokinetics (Cmax and the nadir) previously reported in vivo. METHODS: Diverse cellular and biochemical functions were monitored, which cumulatively can provide insights into some vital cellular functions. Cell proliferation and migration were assessed by quantitative microscopy. Mitochondrial metabolic functions were tested by the MTT assay, and cytosolic esterase activity was studied by the calcein AM assay. Chaperons (CHOP), BiP and apoptosis (caspase-3 activation) were monitored by using Western blot (WB). MAPK Erk1/2 signaling was assessed by the detection of the phosphorylated form of the protein (P-Erk 1/2) and its translocation into the cell nucleus. RESULTS: At all concentrations tested (0.001-0.1mg/mL), FDP-NV did not affect any of the biomarkers of cell integrity of HepG2 cells. In contrast, the proliferation of HUVEC was affected at the highest concentration tested (0.1mg/mL, Cmax). Exposure of HUVEC to (0.01 mg/mL) FDP-NV had a mild-moderate effect on cell proliferation as evident in the MTT assay and was absent when proliferation was assessed by direct cell counting or by using the calcein AM assays. In both cell types, exposure to the highest concentration (0.1 mg/mL) of FDP-NV did neither affect FBS-stimulated cell signaling (MAPK Erk1/2 phosphorylation) nor did it activate of Caspase 3. CONCLUSION: Our data suggest that FDP-NV-800nm are largely biocompatible with HepG-2 cells proliferation within the pharmacokinetic data reported previously. In contrast, HUVEC proliferation at the highest exposure dose (0.1 mg/mL) responded adversely with respect to several biomarkers of cell integrity. However, since the Cmax levels are very short-living, the risk for endothelial injury is likely minimal for slow rate cell proliferation such as endothelial cells.