angiogenin and Neoplasms

For many years, epidemiological studies have suggested an association between cancer and neurodegenerative disorders-two disease processes that seemingly have little in common. Although these two disease processes share disruptions in a wide range of cellular pathways, including cell survival, cell death and the cell cycle, the end result is very divergent: uncontrolled cell survival and proliferation in cancer and progressive neuronal cell death in neurodegeneration. Despite the clinical data connecting these two disease processes, little is known about the molecular links between them. Among the mechanisms affected in cancer and neurodegenerative diseases, alterations in RNA metabolism are obtaining significant attention given the critical role for RNA transcription, maturation, transport, stability, degradation and translation in normal cellular function. RNA-binding proteins (RBPs) are integral to each stage of RNA metabolism through their participation in the formation of ribonucleoprotein complexes (RNPs). RBPs have a broad range of functions including posttranscriptional regulation of mRNA stability, splicing, editing and translation, mRNA export and localization, mRNA polyadenylation and miRNA biogenesis, ultimately impacting the expression of every single gene in the cell. In this review, we examine the evidence for RBPs as being key a molecular linkages between cancer and neurodegeneration.

Topics: Adenosine Deaminase; Aged; Aging; DNA-Binding Proteins; ELAV Proteins; Fragile X Mental Retardation Protein; Guanine Nucleotide Exchange Factors; Humans; Neoplasms; Neurodegenerative Diseases; Ribonuclease, Pancreatic; RNA-Binding Protein EWS; RNA-Binding Protein FUS; RNA-Binding Proteins; RNA, Messenger; RNA, Neoplasm; TATA-Binding Protein Associated Factors

Angiogenesis is essential for tumor growth and metastasis. Many signaling pathways are involved in regulating tumor angiogenesis, with the vascular endothelial growth factor pathway being of particular interest. The recognition of the heterogeneity in tumor vasculature has led to better predictions of prognosis through differential analyses of the vasculature. However, the clinical benefits from antiangiogenic therapy are limited, because many antiangiogenic agents cannot provide long-term survival benefits, suggesting the development of drug resistance. Activation of the hypoxia and c-Met pathways, as well as other proangiogenic factors, has been shown to be responsible for such resistance. Vessel co-option could be another important mechanism. For future development, research to improve the efficacy of antiangiogenic therapy includes (a) using tumor-derived endothelial cells for drug screening; (b) developing the drugs focusing on specific tumor types; (c) developing a better preclinical model for drug study; (d) developing more accurate biomarkers for patient selection; (e) targeting the c-Met pathway or other pathways; and (f) optimizing the dose and schedule of antiangiogenic therapy. In summary, the future of antiangiogenic therapy for cancer patients depends on our efforts to develop the right drugs, select the right patients, and optimize the treatment conditions.

Topics: Angiogenesis Inhibitors; Cell Proliferation; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Microvessels; Neoplasm Metastasis; Neoplasms; Neovascularization, Pathologic; Proto-Oncogene Proteins c-met; Ribonuclease, Pancreatic; Signal Transduction; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factors

Metastatic, rather than primary tumours are responsible for ninety percent cancer deaths. Despite significant advances in the understanding of molecular and cellular mechanisms in tumour metastases, there are limitations in preventive treatment of metastatic tumours. Much evidence arising from laboratory and clinical studies suggests that growth factors and their receptors are implicated in cancer metastases development. We review the origin and production of growth factors and their receptors in all stages of cancer metastases including epithelial-mesenchymal transition, cancer cell invasion and migration, survival within the circulation, seeding at distant organs and metastatic tumour angiogenesis. The functions of growth factors and their receptors are also discussed. This review presents the efforts made in understanding this challenge to aid in the development of new treatment strategies for cancer metastases.

Topics: Angiopoietins; Animals; Apoptosis; Cell Movement; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Glucose-6-Phosphate Isomerase; Hepatocyte Growth Factor; Humans; Insulin-Like Growth Factor I; Intercellular Signaling Peptides and Proteins; Interleukin-8; Multienzyme Complexes; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Seeding; Neoplasms; Neoplastic Cells, Circulating; Neovascularization, Pathologic; Phosphodiesterase I; Phosphoric Diester Hydrolases; Pyrophosphatases; Receptor, IGF Type 1; Receptors, Growth Factor; Ribonuclease, Pancreatic; Smad Proteins; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Angiogenesis is an important mediator of tumor progression. As tumors expand, diffusion distances from the existing vascular supply increases resulting in hypoxia. Sustained expansion of a tumor mass requires new blood vessel formation to provide rapidly proliferating tumor cells with an adequate supply of oxygen and metabolites. The key regulator of hypoxia-induced angiogenesis is the transcription factor hypoxia inducible factor (HIF)-1. Multiple HIF-1 target genes have been shown to modulate angiogenesis by promoting the mitogenic and migratory activities of endothelial cells. Because of this, hypoxia-induced angiogenesis has become an attractive target for cancer therapy, however the mechanisms involved during this process and how best to target it for cancer therapy are still under investigation. This review will cover the current understanding of hypoxia-induced tumor angiogenesis and discuss the caveats of hypoxia-targeted antiangiogenic therapy for the treatment of cancer.

Topics: Angiogenesis Inducing Agents; Angiogenesis Inhibitors; Cell Division; Cell Hypoxia; Humans; Neoplasms; Neovascularization, Pathologic; Nitric Oxide Synthase; Ribonuclease, Pancreatic; Vascular Endothelial Growth Factor A

Angiogenin is a member of the ribonuclease (RNase) superfamily: enzymes of innate substrate specificity, but divergent functional capacities. Angiogenin is a normal constituent of the circulation and contained in a vasculature that rarely undergoes proliferation, but in some physiological and pathological conditions its levels increase in blood, promoting neovascularization. Hence, angiogenesis is a common pathophysiological attribute of angiogenin. In malignant disease, the most studied pathological state in regard to angionenin, abnormally high levels are seen, which may be of prognostic significance. Angiogenin has also been studied in other non-malignant pathological states. The aim of this review article is to provide an overview of the biochemistry and physiology of angiogenin, specifically in relation to the human pathological states where angiogenin has been implicated and finally, its potential clinical applications.

Topics: Female; Humans; Male; Neoplasms; Neovascularization, Pathologic; Pregnancy; Prognosis; Ribonuclease, Pancreatic

Angiogenin is one of the most potent inducers of neovascularization in experimental models in vivo. Angiogenin is normally present in plasma but overexpressed in cancer patients. The possible involvement of angiogenin in the development of cancer is suggested by its overexpression in patients with a variety of tumours and the observation that angiogenin antagonists prevent the growth of human tumour xenografts in athymic mice. This 14.1-kDa protein has 35% amino acid sequence identity with human pancreatic ribonuclease and displays ribonucleolytic activity. As only angiogenin is able to induce angiogenesis, its biological activities are thought to result from structural characteristics. Although the structural characteristics of angiogenin have been extensively studied, the understanding of its physiological role and of how its properties are expressed is still to be deciphered. This article reviews some of the biological, biochemical and structural properties of angiogenin.

Topics: Angiogenesis Inducing Agents; Animals; Gene Expression; Humans; Neoplasm Proteins; Neoplasms; Neovascularization, Pathologic; Ribonuclease, Pancreatic

Cisplatin (CisPt), a platinum-based chemotherapeutic widely used in the treatment of various cancers, has multiple mechanisms of action, including nuclear DNA (nDNA) and mitochondrial DNA (mDNA) damage and cytoskeletal perturbations affecting, in turn, the membrane transporter activity. CisPt binding to proteins and enzymes may modulate its biochemical mechanism of action and is associated with cancer cell resistance to the drug. In this work, we investigate the interaction between cisplatin and angiogenin (Ang), a protein strongly expressed in many types of cancer and a potent angiogenic factor. The adduct formed upon reaction of CisPt with Ang (Ang@CisPt) was characterized by X-ray crystallography to evidence the exact platination site and by UV-visible (UV-vis) absorption and circular dichroism (CD) spectroscopies to shed light on any possible change in the protein conformation. Furthermore, high-resolution electrospray ionization (ESI) mass spectrometry was utilized to evaluate the Ang : CisPt stoichiometry of the Ang@CisPt adduct. The effect of the Ang@CisPt adduct on a prostate cancer cell line (PC-3) was tested by colorimetric assays in terms of cell viability, at both levels of nuclear and mitochondrial damage, and reactive oxygen species (ROS) production. Cellular imaging by laser scanning confocal microscopy (LSM) was utilized to scrutinize the cytoskeleton actin reorganization and the lysosome and mitochondria organelle perturbation. These studies highlight the possibility of new molecular pathways and targets for CisPt activity.

Topics: Antineoplastic Agents; Cell Line; Cisplatin; Humans; Male; Neoplasms; Ribonuclease, Pancreatic

Transfer RNA is heavily modified and plays a central role in protein synthesis and cellular functions. Here we demonstrate that ALKBH3 is a 1-methyladenosine (m1A) and 3-methylcytidine (m3C) demethylase of tRNA. ALKBH3 can promote cancer cell proliferation, migration and invasion. In vivo study confirms the regulation effects of ALKBH3 on growth of tumor xenograft. The m1A demethylated tRNA is more sensitive to angiogenin (ANG) cleavage, followed by generating tRNA-derived small RNAs (tDRs) around the anticodon regions. tDRs are conserved among species, which strengthen the ribosome assembly and prevent apoptosis triggered by cytochrome c (Cyt c). Our discovery opens a potential and novel paradigm of tRNA demethylase, which regulates biological functions via generation of tDRs.

Topics: Adenosine; AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase; Animals; Apoptosis; Cell Movement; Cell Proliferation; Cytidine; Disease Progression; HeLa Cells; Humans; Mice; Neoplasm Invasiveness; Neoplasms; Ribonuclease, Pancreatic; RNA, Transfer; Xenograft Model Antitumor Assays

Tumor angiogenesis is essential for tumor growth and metastasis and is dependent on key angiogenic factors. Angiogenin (ANG), a 14.2-kDa polypeptide member of the RNase A superfamily, is an angiogenic protein that has been reported to be upregulated and associated with poor prognosis in some human cancers. The mechanisms through which aberrant ANG levels promote specific steps in tumor progression are unknown. Here, we show that ANG expression in human tissues is strongly correlated with an invasive cancer phenotype. We also show that ANG induces cellular survival, proliferation, endothelial tube formation and xenograft angiogenesis and growth. Novel mechanistic investigations revealed that ANG expression stimulated matrix metallopeptidase-2 (MMP2) expression through the phosphorylation of ERK1/2. Targeting ANG in vivo with N65828, a small-molecule inhibitor of the ribonucleolytic activity of human ANG, resulted in the diminution of xenograft tumoral growth through the inhibition of angiogenesis. Our findings support an unrecognized interplay between ANG, ERK1/2 and MMP2 that can impact tumor growth and progression. The targeting of ANG and associated factors could provide a novel strategy to inhibit tumor establishment and growth.

Topics: Animals; Cell Line, Tumor; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Heterografts; Humans; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Mice; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Transplantation; Neoplasms; Neovascularization, Pathologic; Ribonuclease, Pancreatic

Herein, a label-free and highly sensitive electrochemical aptasensor for the detection of angiogenin was proposed based on a conformational change of aptamer and amplification by poly(diallyldimethyl ammonium chloride) (PDDA)-functionalized graphene/gold nanoparticles (AuNPs) composites-modified electrode. PDDA-functionalized graphene (P-GR) nanosheets as the building block in the self-assembly of GR nanosheets/AuNPs heterostructure enhanced the electrochemical detection performance. The electrochemical aptasensor has an extraordinarily sensitive response to angiogenin in a linear range from 0.1pM to 5nM with a detection limit of 0.064pM. The developed sensor provides a promising strategy for the cancer diagnosis in medical application in the future.

Topics: Allyl Compounds; Aptamers, Nucleotide; Biosensing Techniques; Electrochemical Techniques; Equipment Design; Gold; Graphite; Humans; Limit of Detection; Metal Nanoparticles; Nanocomposites; Neoplasms; Polyamines; Polyelectrolytes; Quaternary Ammonium Compounds; Ribonuclease, Pancreatic

Human RNASET2 has been implicated in antitumorigenic and antiangiogenic activities, independent of its ribonuclease capacities. We constructed a truncated version of human RNASET2, starting at E50 (trT2-50) and devoid of ribonuclease activity. trT2-50 maintained its ability to bind actin and to inhibit angiogenesis and tumorigenesis. trT2-50 binds to cell surface actin and formed a complex with actin in vitro. The antiangiogenic effect of this protein was demonstrated in human umbilical vein endothelial cells (HUVECs) by its ability to arrest tube formation on Matrigel, induced by angiogenic factors. Immunofluorescence staining of HUVECs showed nuclear and cytosolic RNASET2 protein that was no longer detectable inside the cell following trT2-50 treatment. This effect was associated with disruption of the intracellular actin network. trT2-50 co-localized with angiogenin, suggesting that both molecules bind (or compete) for similar cellular epitopes. Moreover, trT2-50 led to a significant inhibition of tumor development. Histological analysis demonstrated abundant necrotic tissue and a substantial loss of endothelial structure in trT2-50-treated tumors. Collectively, the present results indicate that trT2-50, a molecule engineered to be deficient of its catalytic activity, still maintained its actin binding and anticancer-related biological activities. We therefore suggest that trT2-50 may serve as a potential cancer therapeutic agent.

Topics: Actins; Amino Acid Sequence; Animals; Antineoplastic Agents; Carcinogenesis; Cell Line, Tumor; Cell Membrane; Cell Nucleus; Chromatography, Affinity; Cytosol; Epitopes; Female; Glycosylation; Heparitin Sulfate; Human Umbilical Vein Endothelial Cells; Humans; Mice; Mice, Nude; Molecular Sequence Data; Neoplasm Transplantation; Neoplasms; Neovascularization, Pathologic; Protein Denaturation; Protein Folding; Recombinant Proteins; Ribonuclease, Pancreatic; Ribonucleases; Tumor Suppressor Proteins

Topics: Animals; Aptamers, Nucleotide; Base Sequence; Biological Transport; Cell Line, Tumor; Chlorocebus aethiops; Chlorophyllides; COS Cells; HeLa Cells; Humans; Neoplasms; Photochemotherapy; Photosensitizing Agents; Porphyrins; Ribonuclease, Pancreatic

ANG is a plasma protein with angiogenic and ribonucleolytic activity implicated in tumor growth, heart failure, wound healing, asthma, and the composition of the adult gut microflora. Human mast cells (HuMC) are similarly associated with modulation of vascular permeability, angiogenic processes, wound healing, and asthma. We hypothesized that HuMC express and secrete ANG in response to divergent stimuli. ANG expression was evaluated in the LAD2 HMC, the HMC-1, and CD34+-derived HuMC, following exposure to live Escherichia coli, TLR ligands, or neuropeptides and following FcepsilonRI aggregation. Expression and production of ANG were determined by microarray analysis, qRT-PCR, confocal microscopy, and ELISA. Microarray analysis showed that ANG is up-regulated by LAD2 cells exposed to live E. coli. qRT-PCR analysis revealed that LAD2, HMC-1, and HuMC constitutively expressed ANG mRNA and that it was up-regulated by exposure to E. coli. Activation of HuMC by FcepsilonRI aggregation resulted in release of small amounts of ANG (<100 pg/mL), whereas compound 48/80, NGF, LPS, PGN, and flagellin activated HuMC to secrete >160 pg/mL ANG. These observations demonstrate that HuMC store and secrete ANG to a variety of stimuli and suggest that MC-derived ANG is available in the subsequent inflammatory response.

Topics: Animals; Asthma; Chickens; Escherichia coli; Flow Cytometry; Humans; Inflammation; Mast Cells; Neoplasms; Neovascularization, Physiologic; Oligonucleotide Array Sequence Analysis; Ribonuclease, Pancreatic; Wound Healing

The most potent immunotoxins (ITs) developed to date contain bacterial or plant cytotoxic components. As these are potentially immunogenic in man, human proteins are preferred for the long-term treatment of cancer. We have developed the first humanized IT for the treatment of CD64 malignancies such as acute myeloid leukemia. The bacterially expressed IT is composed of a humanized anti-CD64 single chain fragment [h22(scFv)] genetically fused to the human RNase angiogenin. As angiogenin lacks a dedicated translocation domain responsible for the higher potency of bacterial and plant-derived toxins, we have incorporated a recombinant adapter that contains a synthetic translocation domain flanked by proteolytically cleavable endosomal and cytosolic consensus sites. Although insertion of the adapter increased the cytotoxicity by up to 20-fold, serum stability was markedly reduced. Therefore, we designed a modified adapter variant with the endosomal-cleavable peptide deleted. The IT containing the truncated adapter showed significantly higher cytotoxicity than the adapter-free IT and superior serum stability to facilitate the potential applications in patients.

Topics: Cell Proliferation; Cytotoxicity, Immunologic; Epitopes; Genetic Engineering; Humans; Immunoglobulin Fragments; Immunotherapy; Immunotoxins; Membrane Transport Modulators; Neoplasms; Protein Binding; Receptors, IgG; Recombinant Fusion Proteins; Ribonuclease, Pancreatic; U937 Cells

We have previously shown that the aminoglycoside antibiotic neomycin blocks the nuclear translocation of angiogenin and inhibits its angiogenic activity. However, neomycin has not been considered as a favorable drug candidate for clinical development because of its known nephrotoxicity and ototoxicity. The aim of this study is to determine whether neamine, a nontoxic derivative of neomycin, possesses antitumor activity.. The effect of neamine on the nuclear translocation of angiogenin was examined by means of immunofluorescence and Western blotting. The antitumor activity of neamine was determined with three different animal models.. Neamine effectively blocked the nuclear translocation of angiogenin in endothelial cells and inhibited angiogenin-induced cell proliferation. It inhibited the establishment of human tumor xenografts in athymic mice in both ectopic and orthotopic tumor models. It also inhibited the progression of established human tumor transplants, whereas the structurally related antibiotic paromomycin had no effect. Immunohistochemical staining showed that both angiogenesis and cancer cell proliferation are inhibited by neamine.. These results suggest that the nontoxic aminoglycoside antibiotic neamine is an effective inhibitor of nuclear translocation of angiogenin and may serve as an inhibitor for angiogenin-induced angiogenesis and cancer progression.

Topics: Active Transport, Cell Nucleus; Aminoglycosides; Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Cell Nucleus; Cell Proliferation; Endothelial Cells; Framycetin; Humans; Mice; Mice, Nude; Neoplasms; Neovascularization, Pathologic; Ribonuclease, Pancreatic; Umbilical Veins; Xenograft Model Antitumor Assays

The purpose of this study is to develop a high-throughput approach to detect protein expression from hundreds and thousands of samples and to apply this technology to profile circulating angiogenic factor protein levels in patients with gynecological tumors.. Analytes containing a mixture of protein are immobilized onto antibody-coated surface of support in array format. The presence of protein in analytes is detected with biotin-labeled antibody coupled with an enhanced chemiluminescence or fluorescence detection system. The exact amount of protein can be quantitatively measured. The expression levels of five angiogenic factors (angiogenin, interleukin 8, vascular endothelial growth factor, platelet-derived growth factor, and epidermal growth factor) from 157 samples were quantitatively measured using this novel protein array technology and were statistically analyzed. The expression patterns of angiogenic factors were analyzed using two-way hierarchical cluster analysis approach.. A novel protein array technology, which can simultaneously and quantitatively measure few protein levels from hundreds and thousands of samples was developed. Only minute amounts of sample are required for the assay. This approach also features high sensitivity and specificity. Using this novel protein array approach, we analyzed the plasma expression levels of five angiogenic factors in 137 patients diagnosed with a tumor and 20 controls. Statistical analysis reveals different expression levels of angiogenic factors between patients and controls. Cluster analysis suggests a possible classification of normal subjects from patients.. Enhanced protein profiling arrays provide a high-throughput and sensitive system to detect one or few protein from hundreds and thousands of samples. Such an approach should have broad application in biomedical discovery.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Biotin; Cell Line, Tumor; Cluster Analysis; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Female; Genital Neoplasms, Female; Humans; Immunoglobulin G; Interleukin-8; Luminescent Measurements; Microscopy, Fluorescence; Middle Aged; Multigene Family; Neoplasms; Neovascularization, Pathologic; Oligonucleotide Array Sequence Analysis; Platelet-Derived Growth Factor; Protein Array Analysis; Ribonuclease, Pancreatic; Sensitivity and Specificity; Tissue Distribution; Vascular Endothelial Growth Factor A

Apolipoprotein(a) (apo(a)) contains tandemly repeated kringle domains that are closely related to plasminogen kringle 4, followed by a single kringle 5-like domain and an inactive protease-like domain. Recently, the anti-angiogenic activities of apo(a) have been demonstrated both in vitro and in vivo. However, its effects on tumor angiogenesis and the underlying mechanisms involved have not been fully elucidated. To evaluate the anti-angiogenic and anti-tumor activities of the apo(a) kringle domains and to elucidate their mechanism of action, we expressed the last three kringle domains of apo(a), KIV-9, KIV-10, and KV, in Escherichia coli. The resultant recombinant protein, termed rhLK68, exhibited a dose-dependent inhibition of basic fibroblast growth factor-stimulated human umbilical vein endothelial cell proliferation and migration in vitro and inhibited the neovascularization in chick chorioallantoic membranes in vivo. The ability of rhLK68 to abrogate the activation of extracellular signal-regulated kinases appears to be responsible for rhLK68-mediated anti-angiogenesis. Furthermore, systemic administration of rhLK68 suppressed human lung (A549) and colon (HCT-15) tumor growth in nude mice. Immunohistochemical examination and in situ hybridization analysis of the tumors showed a significant decrease in the number of blood vessels and the reduced expression of vascular endothelial growth factor, basic fibroblast growth factor, and angiogenin, indicating that suppression of angiogenesis may have played a significant role in the inhibition of tumor growth. Collectively, these results suggest that a truncated apo(a), rhLK68, is a potent anti-angiogenic and anti-tumor molecule.

Topics: Allantois; Animals; Apolipoproteins A; Cell Division; Cell Movement; Cells, Cultured; Chickens; Chorion; Colonic Neoplasms; Dose-Response Relationship, Drug; Endothelial Growth Factors; Endothelium, Vascular; Escherichia coli; Female; Fibroblast Growth Factor 2; Gene Expression; Humans; In Situ Hybridization; Intercellular Signaling Peptides and Proteins; Kringles; Lung Neoplasms; Lymphokines; Mice; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neoplasm Transplantation; Neoplasms; Neovascularization, Pathologic; Neovascularization, Physiologic; Peptide Fragments; Phosphorylation; Recombinant Proteins; Ribonuclease, Pancreatic; RNA, Messenger; Tumor Cells, Cultured; Umbilical Veins; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The murine monoclonal antibody 26-2F neutralizes the angiogenic and ribonucleolytic activities of human angiogenin (ANG) and is highly effective in preventing the establishment and metastatic dissemination of human tumors in athymic mice. Here we report a 2.0 A resolution crystal structure for the complex of ANG with the Fab fragment of 26-2F that reveals the detailed interactions between ANG and the complementarity-determining regions (CDRs) of the antibody. Surprisingly, Fab binding induces a dramatic conformational change in the cell binding region of ANG at the opposite end of the molecule from the combining site; crosslinking experiments indicate that this rearrangement also occurs in solution. The ANG-Fab complex structure should be invaluable for designing maximally humanized versions of 26-2F for potential clinical use.

Topics: Amino Acid Sequence; Complementarity Determining Regions; Epitope Mapping; Humans; Immunoglobulin Fab Fragments; Molecular Sequence Data; Neoplasms; Neutralization Tests; Protein Conformation; Ribonuclease, Pancreatic

The results of previous preclinical and clinical studies have identified angiogenin (ANG) as a potentially important target for anticancer therapy. Here we report the design and implementation of a high-throughput screening assay to identify small molecules that bind to the ribonucleolytic active site of ANG, which is critically involved in the induction of angiogenesis by this protein. Screening of 18,310 compounds from the National Cancer Institute (NCI) Diversity Set and ChemBridge DIVERSet yielded 15 hits that inhibit the enzymatic activity of ANG with K(i) values <100 microM. One of these, NCI compound 65828 [8-amino-5-(4'-hydroxybiphenyl-4-ylazo)naphthalene-2-sulfonate; K(i) = 81 microM], was selected for more detailed studies. Minor changes in ANG or ligand structure markedly reduced potency, demonstrating that inhibition reflects active-site rather than nonspecific binding; these observations are consistent with a computationally generated model of the ANG.65828 complex. Local treatment with modest doses of 65828 significantly delayed the formation of s.c. tumors from two distinct human cancer cell types in athymic mice. ANG is the likely target involved because (i) a 65828 analogue with much lower potency against the enzymatic activity of ANG failed to exert any antitumor effect, (ii) tumors from 65828-treated mice had fewer interior blood vessels than those from control mice, and (iii) 65828 appears to have no direct effect on the tumor cells. Our findings provide considerable support for the targeting of the enzymatic active site of ANG as a strategy for developing new anticancer drugs.

Topics: Amino Acid Substitution; Animals; Anticarcinogenic Agents; Cell Line; Drug Evaluation, Preclinical; Genetic Variation; Humans; Kinetics; Mice; Mice, Nude; Molecular Structure; Naphthalenesulfonates; Neoplasms; Neovascularization, Pathologic; Recombinant Proteins; Reproducibility of Results; Ribonuclease, Pancreatic; Structure-Activity Relationship; Transplantation, Heterologous; Tumor Cells, Cultured

Human angiogenin (Ang), an unusual member of the pancreatic RNase superfamily, is a potent inducer of angiogenesis in vivo. Its ribonucleolytic activity is weak (10(4) to 10(6)-fold lower than that of bovine RNase A), but nonetheless seems to be essential for biological function. Ang has been implicated in the establishment of a wide range of human tumours and has therefore emerged as an important target for the design of new anti-cancer compounds. We report high-resolution crystal structures for native Ang in two different forms (Pyr1 at 1.8 A and Met-1 at 2.0 A resolution) and for two active-site variants, K40Q and H13A, at 2.0 A resolution. The native structures, together with earlier mutational and biochemical data, provide a basis for understanding the unique functional properties of this molecule. The major structural features that underlie the weakness of angiogenin's RNase activity include: (i) the obstruction of the pyrimidine-binding site by Gln117; (ii) the existence of a hydrogen bond between Thr44 and Thr80 that further suppresses the effectiveness of the pyrimidine site; (iii) the absence of a counterpart for the His119-Asp121 hydrogen bond that potentiates catalysis in RNase A (the corresponding aspartate in Ang, Asp116, has been recruited to stabilise the blockage of the pyrimidine site); and (iv) the absence of any precise structural counterparts for two important purine-binding residues of RNase A. Analysis of the native structures has revealed details of the cell-binding region and nuclear localisation signal of Ang that are critical for angiogenicity. The cell-binding site differs dramatically from the corresponding regions of RNase A and two other homologues, eosinophil-derived neurotoxin and onconase, all of which lack angiogenic activity. Determination of the structures of the catalytically inactive variants K40Q and H13A has now allowed a rigorous assessment of the relationship between the ribonucleolytic and biological activities of Ang. No significant change outside the enzymatic active site was observed in K40Q, establishing that the loss of angiogenic activity for this derivative is directly attributable to disruption of the catalytic apparatus. The H13A structure shows some changes beyond the ribonucleolytic site, but sites involved in cell-binding and nuclear translocation are essentially unaffected by the amino acid replacement.

Topics: Amino Acid Sequence; Binding Sites; Crystallography, X-Ray; Egg Proteins; Eosinophil-Derived Neurotoxin; Humans; Hydrogen Bonding; Models, Molecular; Molecular Sequence Data; Mutation; Neoplasms; Neovascularization, Pathologic; Protein Structure, Secondary; Protein Structure, Tertiary; Proteins; Ribonuclease, Pancreatic; Ribonucleases; Sequence Alignment

Topics: Angiogenesis Inducing Agents; Animals; Growth Substances; Humans; Neoplasm Proteins; Neoplasms; Rats; Ribonuclease, Pancreatic

Topics: Amino Acid Sequence; Angiogenesis Inducing Agents; Animals; Growth Substances; Humans; Neoplasm Proteins; Neoplasms; Rats; Ribonuclease, Pancreatic