RESUMEN
Angiostatin is a potent inhibitor of angiogenesis generated in cancer-bearing hosts by tumor-derived proteases. Because the naturally occurring bone and prostate cancers of pet dogs provide unique model systems to study factors that regulate cancer progression and tumor dormancy, we investigated the capacity of these tumors to generate angiostatin. We determined that angiostatin fragments are present in urine of dogs with bone cancer. The identity of these fragments was confirmed by comparison of the experimentally determined protein sequence to that of a clone of canine angiostatin. Importantly, these fragments were absent in urine collected from the same dogs after complete surgical removal of the primary tumor. We also demonstrate that canine prostate cancer cells are capable of processing plasminogen to angiostatin in vitro. These findings provide rationale for using spontaneous canine tumor models to isolate endogenous angiogenesis inhibitors and to investigate their therapeutic use against cancer.
Asunto(s)
Neoplasias Óseas/veterinaria , Enfermedades de los Perros/metabolismo , Osteosarcoma/veterinaria , Fragmentos de Péptidos/metabolismo , Plasminógeno/metabolismo , Neoplasias de la Próstata/veterinaria , Angiostatinas , Animales , Especificidad de Anticuerpos , Neoplasias Óseas/metabolismo , Bovinos , División Celular/efectos de los fármacos , Progresión de la Enfermedad , Perros , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Masculino , Datos de Secuencia Molecular , Osteosarcoma/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/orina , Plasminógeno/química , Plasminógeno/genética , Plasminógeno/orina , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Homología de Secuencia de Aminoácido , Células Tumorales CultivadasRESUMEN
A primary inoculum of human pancreatic cancer cells (BxPC-3) has the ability to inhibit the growth of a secondary tumor in an in vivo animal model. Such ability suggests that the primary tumor is producing inhibitors that act at the site of the secondary tumor. Accordingly we attempted to discover which inhibitors are produced by pancreatic cancer cells. We determined that pancreatic cancer cells process angiostatin isoforms from plasminogen. Additionally, we isolated and characterized an uncleaved "latent" antiangiogenic antithrombin (aaAT) molecule processed from systemically available AT by pancreatic cancer cells as well as a cleaved form of aaAT processed from systemically available AT by pancreatic cancer cells. Human AT, cleaved with human neutrophil elastase, inhibits angiogenesis in the chorioallantoic membrane assay. This human aaAT molecule is able to inhibit the growth of pancreatic tumors in immune-compromised mice. Our work represents the first demonstration of multiple angiogenesis inhibitors from a single tumor and suggests that antiangiogenic therapies may provide an avenue for future treatment of pancreatic cancer.
Asunto(s)
Adenocarcinoma/metabolismo , Inhibidores de la Angiogénesis/biosíntesis , Antitrombinas/biosíntesis , Neovascularización Patológica/prevención & control , Neoplasias Pancreáticas/metabolismo , Fragmentos de Péptidos/biosíntesis , Plasminógeno/biosíntesis , Adenocarcinoma/sangre , Adenocarcinoma/irrigación sanguínea , Inhibidores de la Angiogénesis/aislamiento & purificación , Inhibidores de la Angiogénesis/farmacología , Angiostatinas , Animales , Antitrombinas/aislamiento & purificación , Antitrombinas/farmacología , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Carcinoma Pulmonar de Lewis/patología , División Celular/fisiología , Embrión de Pollo , Medios de Cultivo Condicionados , Endotelio Vascular/citología , Fibrosarcoma/irrigación sanguínea , Fibrosarcoma/patología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Neovascularización Fisiológica/efectos de los fármacos , Neoplasias Pancreáticas/sangre , Neoplasias Pancreáticas/irrigación sanguínea , Plasminógeno/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In the first Phase I clinical trials of endostatin as an antiangiogenic therapy for cancer, the protein was administered as an i.v. bolus for approximately 20-30 min each day. This protocol was based on experimental studies in which animals were treated by s.c. bolus once a day. However, it was not clear in the previous studies whether this schedule could be maximized further. Therefore, we developed experimental models involving continuous administration of endostatin to determine the potency and efficacy of this approach. Endostatin was administered to tumor-bearing mice either s.c. or i.p. in single bolus doses. The efficacy of these regimens was compared with endostatin administered continuously via an i.p. implanted mini-osmotic pump. Our results show that endostatin remains stable and active in mini-osmotic pumps for at least 7 days. We show that endostatin injected i.p. is rapidly cleared within 2 h, whereas endostatin administered continuously via mini-osmotic pump maintains systemic concentrations of 200-300 ng/ml for the duration of administration. Furthermore, continuous i.p. administration of endostatin results in more effective tumor suppression at significantly reduced doses (5-fold), compared with bolus administration. Additional experiments using a human pancreatic cancer model in severe combined immunodeficient mice showed that there was a significant decrease in the microvessel density between the treatment groups and the control group. These data show that continuous administration of human endostatin results in sustained systemic concentrations of the protein leading to: (a) increased efficacy manifested as increased tumor regression; and (b) an 8-10-fold decrease in the dose required to achieve the same antitumor effect as the single daily bolus administration of endostatin. On the basis of this approach, an additional clinical trial has been designed and initiated and is under way in two countries.
Asunto(s)
Inhibidores de la Angiogénesis/administración & dosificación , Antineoplásicos/administración & dosificación , Colágeno/administración & dosificación , Fragmentos de Péptidos/administración & dosificación , Animales , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Colágeno/farmacocinética , Estabilidad de Medicamentos , Endostatinas , Fibrosarcoma/irrigación sanguínea , Fibrosarcoma/tratamiento farmacológico , Humanos , Bombas de Infusión Implantables , Infusiones Parenterales , Inyecciones Intraperitoneales , Masculino , Ratones , Ratones Endogámicos CBA , Ratones SCID , Neovascularización Patológica/tratamiento farmacológico , Presión Osmótica , Neoplasias Pancreáticas/irrigación sanguínea , Neoplasias Pancreáticas/tratamiento farmacológico , Fragmentos de Péptidos/farmacocinética , Ensayos Antitumor por Modelo de XenoinjertoAsunto(s)
Antineoplásicos/metabolismo , Neoplasias Experimentales/tratamiento farmacológico , Fragmentos de Péptidos/fisiología , Plasminógeno/fisiología , Polisacáridos/fisiología , Angiostatinas , Animales , Antineoplásicos/química , Glicosilación , Humanos , Ratones , Estructura Molecular , Neoplasias Experimentales/metabolismo , Fragmentos de Péptidos/química , Plasminógeno/química , Polisacáridos/químicaRESUMEN
Human plasminogen, the inactive precursor of plasmin, exists in two major glycoforms. Plasminogen 1 contains an N-linked oligosaccharide at Asn-289 and an O-linked oligosaccharide at Thr-345. Plasminogen 2 is known to contain only an O-linked oligosaccharide at Thr-345. However, plasminogen 2 displays a further well documented microheterogeneity dependent on the N-acetylneuraminic acid content, which has functional consequences with regard to activation of plasminogen. The proposed structure and number of known oligosaccharide linkages in plasminogen 2 is insufficient to account for this microheterogeneity. In the present study, a combination of trypsin digestion, lectin affinity chromatography, Edman degradation amino acid sequence analysis, carbohydrate composition analysis, and mass spectrometry revealed the existence of a novel site for O-linked glycosylation on plasminogen 2 at Ser-248. Direct evidence for the structure of the carbohydrate was obtained from a combination of lectin affinity chromatography, desialylation experiments, and mass spectrometry analysis. These findings provide a structural basis for some of the observed microheterogeneity, and have implications with regard to the known functional consequences of the extent of sialylation of plasminogen.
Asunto(s)
Plasminógeno/química , Trisacáridos/química , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Glicosilación , Humanos , Datos de Secuencia MolecularAsunto(s)
Fraude/legislación & jurisprudencia , Medicare , Revelación de la Verdad , Humanos , Estados UnidosRESUMEN
Six glycoforms of plasminogen 2 were isolated using a combination of lectin affinity chromatography and chromatofocussing, and the sialic acid content of each glycoform was determined. The kinetics of activation of each glycoform by tissue-type plasminogen activator were analyzed on a fibrin surface and in solution. The second-order rate constant (measured on a fibrin surface) decreased from 1.65 x 10(6) M-1 s-1 to 3.77 x 10(4) M-1 s-1 as the sialic acid content of the glycoforms increased from 1.3 mol/mol of protein to 13.65 mol/mol of protein. A similar correlation was noted for activation in solution. Each glycoform was converted to plasmin, and the inhibition constants for the reaction between alpha 2-antiplasmin and plasmin glycoforms were determined. All overall Ki values, reflecting the final essentially irreversible complex, were in the picomolar range. Sialic acid does not affect inhibition of plasmin by alpha 2-antiplasmin; however, hypersialylated plasmin does not appear to have a kringle-dependent component to inhibition.
Asunto(s)
Isoenzimas/química , Plasminógeno/química , Ácidos Siálicos/análisis , Cromatografía de Afinidad , Fibrinólisis , Glicosilación , Homeostasis , Humanos , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Cinética , Plasminógeno/aislamiento & purificación , Plasminógeno/metabolismo , Activador de Tejido Plasminógeno/metabolismoRESUMEN
Five highly soluble, chymotrypsin-like, neutral serine proteases, with molecular masses in the range 30-33 kDa, were isolated from Trichinella spiralis-infected mouse small intestine. These enzymes were closely related antigenically on Western blotting and by Ouchterlony double diffusion using a polyclonal, cross-absorbed, sheep antibody raised against mouse mast cell protease-1 (MMCP-1) and on the basis of N-terminal amino acid sequence analysis, were identified as variant forms of MMCP-1. Substrate and inhibitor analysis confirmed that the five variants (MMCP-1 A-E) had similar characteristics, although highly significant (P = 0.025 to P < 0.0001) variations in Km and kcat, were detected. Against human alpha 1-proteinase inhibitor the Ki for MMCP-1C (45 pM) was significantly (P < 0.0001) greater than those for the other proteases (0.76-2.2 pM). The differences in electrophoretic mobility are probably a result of variable glycosylation, since removal of N-linked carbohydrate produced a polypeptide of approx. 28 kDa in each case which was, like the native enzyme, immunoreactive on Western blotting. A much less soluble 28 kDa enzyme was isolated from serosal mast cells and identified as MMCP-4 by N-terminal amino acid sequencing. Like MMCP-1 it has chymotrypsin-like substrate specificities with activity at neutral pH. However, it was antigenically distinct from MMCP-1 and, using sheep anti-MMCP-1, was not detected on Western blotting or by Ouchterlony double diffusion, e.l.i.s.a. or immunohistochemistry. This last technique established that the MMCP-1 variants were uniquely present in enteric mast cells, thereby providing a highly selective means of distinguishing the mucosal and connective tissue mast cell subsets in the mouse.
Asunto(s)
Mastocitos/enzimología , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Autorradiografía , Cromatografía por Intercambio Iónico , Quimasas , Electroforesis en Gel de Poliacrilamida , Femenino , Glicosilación , Intestinos/enzimología , Cinética , Masculino , Ratones , Datos de Secuencia Molecular , Serina Endopeptidasas/química , Serina Endopeptidasas/inmunología , Serina Endopeptidasas/aislamiento & purificación , Especificidad por SustratoRESUMEN
Rat mast cell proteinase II (RMCP II) from mucosal mast cells was titrated into rat serum, and the resulting serine proteinase inhibitor (serpin)-enzyme complex was purified by affinity chromatography on anti-RMCP II-Sepharose 4B and by Mono-Q anion-exchange. The purified complex was used to raise polyclonal antibodies which, after cross-absorption against RMCP II-Sepharose 4B, were specific for serpin and were used to affinity purify two rat serpin molecules (RSI and RSII) that inhibit RMCP II in rat serum. The kinetic constants characterizing the interaction between RMCP II and RSI and RSII are ka, 2.2 x 10(5) and 1.65 x 10(5) M-1 s-1, respectively; Ki, 3.6 x 10(-10) and 1.0 x 10(-9) M; and kd, 7.9 x 10(-5) and 1.65 x 10(-4) s-1. Amino-terminal sequence analysis indicated that RSI and RSII are distinct, differing at the amino-terminal residues, and are products of the rat Spi-1 locus. Rat mast cell proteinase I (RMCP I) from connective tissue mast cells cleaved both RSI and RSII and was not inhibited.
Asunto(s)
Mastocitos/enzimología , Inhibidores de Serina Proteinasa , Serpinas/fisiología , Secuencia de Aminoácidos , Animales , Cromatografía por Intercambio Iónico , Quimasas , Masculino , Datos de Secuencia Molecular , Ratas , Ratas Endogámicas , Serina Endopeptidasas/aislamiento & purificación , Serina Endopeptidasas/metabolismoRESUMEN
The in vitro inhibitory potency of 20 structurally related alkanoic and arylalkanoic acids has been investigated on rat liver UDP-glucuronosyltransferase. These compounds were tested on the microsomal and purified enzyme, and a cloned cDNA expressed in COS 7 cell cultures. Among all the acids tested, 7,7,7-triphenylheptanoic acid was the most powerful inhibitor of bilirubin:UDP-glucuronosyltransferase with a lower effect on 1-naphtol, androsterone and testosterone glucuronidation. The inhibition was competitive towards the microsomal and purified bilirubin:UDP-glucuronosyltransferases with Kiapp values of 12.0 microM and 1.6 microM, respectively. Twenty analogues were examined, and the results showed that their inhibitory potency on bilirubin:UDP-glucuronosyltransferase activity was a function of at least three structural features (a) the presence of a hydrophobic triphenyl moiety; (b) the length of the aliphatic chain and (c) the presence of a carboxylic group. These inhibitors were also tested as possible substrates of UDP-glucuronosyltransferases. The strongest inhibitors were poor substrates of rat liver microsomal UDP-glucuronosyltransferases. However, 7,7,7-triphenylheptanoic acid was actively glucuronidated by purified bilirubin:UDP-glucuronosyltransferase, in contrast to its analogues with decreasing alkyl chain length. In addition, glucuronidation of this molecule was enhanced by clofibrate treatment but could not be detected in Gunn rats, which are deficient in bilirubin:UDP-glucuronosyltransferase, further indicating that the glucuronidation of this compound was catalysed by bilirubin:UDP-glucuronosyltransferase. The results suggest that 7,7,7-triphenylheptanoic acid may be a useful structural probe to investigate the molecular basis of glucuronidation of bilirubin and carboxylic acids.
Asunto(s)
Ácidos Carboxílicos/farmacología , Glucuronosiltransferasa , Hexosiltransferasas/metabolismo , Microsomas Hepáticos/enzimología , Animales , Ácidos Carboxílicos/síntesis química , Cromatografía DEAE-Celulosa , Hexosiltransferasas/antagonistas & inhibidores , Hexosiltransferasas/aislamiento & purificación , Cinética , Masculino , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Ratas , Ratas Endogámicas , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Bilirubin UDP-glucuronosyltransferase (UDPGT) activity in sealed hepatic microsomes from clofibrate-treated rats was highly latent and was fully expressed by disruption of vesicles with detergents. Antibodies raised against purified bilirubin UDPGT were used to study the transmembrane orientation of the protein to provide a molecular understanding of the UDPGT latency. Immunoblot analysis of sealed microsomes, and microsomes after treatment with proteinases, showed that only a small portion of the protein resides on the cytoplasmic side of the microsomal vesicles. Treatment of microsomes with sodium deoxycholate allowed subtilisin and proteinase K to cleave the transferase, causing loss of activity and the release of smaller immunodetectable peptides. Treatment of the purified bilirubin UDPGT with peptide N-glycosidase F indicated that the enzyme was a glycoprotein. A working model of the transmembrane topology of bilirubin UDPGT is described.
Asunto(s)
Retículo Endoplásmico/enzimología , Glucosiltransferasas/metabolismo , Hígado/ultraestructura , Animales , Hígado/enzimología , Microsomas Hepáticos/enzimología , Ratas , Ratas EndogámicasRESUMEN
Ten patients with moderate to advanced periodontal disease were subjected to two similar periodontal surgical procedures. Each patient received either intravenous conscious sedation with local anesthesia or local anesthesia only. The stress-reducing effects of a conscious sedation regimen consisting of pentobarbital, meperidine, and diazepam were evaluated in these patients. Stress was evaluated by monitoring changes in serum cortisol, human growth hormone, and vital signs. Blood samples were obtained at 15- to 30-minute intervals throughout each procedure and were evaluated for serum cortisol and growth hormone. The conscious sedation group had significantly lower serum cortisol levels and lower systolic blood pressure, indicating that the patients having periodontal surgery with conscious sedation experienced reduced stress. Physiologic stability was maintained for each patient, indicating that this conscious sedation regimen can be used to reduce measurable parameters of stress that patients develop during periodontal surgery.
Asunto(s)
Anestesia Dental/métodos , Anestesia Local , Enfermedades Periodontales/cirugía , Medicación Preanestésica , Estrés Fisiológico/terapia , Presión Sanguínea , Diazepam , Hormona del Crecimiento/sangre , Humanos , Hidrocortisona/sangre , Masculino , Meperidina , Pentobarbital , Estrés Fisiológico/sangre , Estrés Fisiológico/fisiopatologíaRESUMEN
The widespread introduction of materials for direct bonding into practices and the numerous reports of a variety of toxic reactions to similar materials prompted a study of the toxicity of six adhesives in an animal model. Thirty-nine hamsters were employed to evaluate responses of skin, oral mucosa, and gingiva, sites of possible contamination by adhesives in clinical use. Animals were treated topically with separate components of adhesives. Adhesives were also allowed to polymerize on and remain in contact with tissues. Test sites were examined grossly and histologically and evaluated for toxic response. The monomer component of one adhesive consistently caused gross irritation and histologic inflammation in twelve animals (p less than 0.001) in which it was used. Other adhesives and components did not have this effect. This adhesive was withdrawn from the market and modified at about the same time these experiments were performed. Similar results have not been observed with the modified product. Clinicans should recognize the potential for reaction to adhesives and realize that products with a toxic potential can reach the market. They should be prepared to change adhesives should reactions to one be observed. They should further recognize that absence of reaction in any test system does not preclude reaction in clinical use.