RESUMEN
Cooperative assemblies of transcription factors (TFs) on target gene enhancers coordinate cell proliferation, fate specification, and differentiation through precise and complicated transcriptional mechanisms. Chemical modifications, such as phosphorylation, of TFs induced by cell signaling further modulate the dynamic cooperativity of TFs. In this study, we found that various Ets1-containing TF-DNA complexes respond differently to calcium-induced phosphorylation of Ets1, which is known to inhibit Ets1-DNA binding. Crystallographic analysis of a complex comprising Ets1, Runx1, and CBFß at the TCRα enhancer revealed that Ets1 acquires robust binding stability in the Runx1 and DNA-complexed state, via allosteric mechanisms. This allows phosphorylated Ets1 to be retained at the TCRα enhancer with Runx1, in contrast to other Ets1 target gene enhancers including mb-1 and stromelysin-1. This study provides a structure-based model for cell-signaling-dependent regulation of target genes, mediated via chemical modification of TFs.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , ADN/metabolismo , Proteína Proto-Oncogénica c-ets-1/metabolismo , Animales , Secuencia de Bases , Línea Celular , Subunidad alfa 2 del Factor de Unión al Sitio Principal/química , Subunidad beta del Factor de Unión al Sitio Principal/química , Cristalografía por Rayos X , ADN/química , Humanos , Ratones , Modelos Moleculares , Fosforilación , Unión Proteica , Conformación Proteica , Proteína Proto-Oncogénica c-ets-1/química , Activación TranscripcionalRESUMEN
Gene transcription is regulated in part through the assembly of multiple transcription factors (TFs) on gene enhancers. To enable examination of the mechanism underlying the formation of these complexes and their response to a phosphorylation signal, two kinds of higher-order TF-DNA assemblies were crystallized composed of an unmodified or phosphorylated Ets1 fragment, a Runx1(L94K) fragment and a CBFß fragment on the T-cell receptor (TCR) α gene enhancer. Within these complexes, the Ets1 and Runx1 fragments contain intrinsically disordered regulatory regions as well as their DNA-binding domains. Crystals of the complex containing unmodified Ets1 belonged to space group P212121, with unit-cell parameters a = 78.7, b = 102.1, c = 195.0â Å, and diffracted X-rays to a resolution of 2.35â Å, and those containing phosphorylated Ets1 belonged to the same space group, with unit-cell parameters a = 78.6, b = 101.7, c = 194.7â Å, and diffracted X-rays to a similar resolution. To facilitate crystallization, a Runx1 residue involved in a hydrophobic patch that was predicted to be engaged in crystal packing based on the previously reported structures of Runx1-containing crystals was mutated.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/química , Elementos de Facilitación Genéticos , Genes Codificadores de la Cadena alfa de los Receptores de Linfocito T , Proteína Proto-Oncogénica c-ets-1/química , Secuencia de Aminoácidos , Animales , Cristalización , ADN/química , Humanos , Sustancias Macromoleculares/química , Ratones , Datos de Secuencia Molecular , Unión ProteicaRESUMEN
Shikonin and beta-hydroxyisovalerylshikonin (beta-HIVS) from Lithospermum erythrorhizon inhibit angiogenesis via inhibition of vascular endothelial growth factor receptors (VEGFR) in an adenosine triphosphate-non-competitive manner, although the underlying molecular mechanism has not been fully understood. In the present study, we found that beta-HIVS inhibited angiogenesis within chicken chorioallantoic membrane approximately threefold more efficiently than shikonin. beta-HIVS also significantly inhibited angiogenesis in two other assays, induced either by Lewis lung carcinoma cells implanted in mouse dorsal skin or by VEGF in s.c. implanted Matrigel plugs and metastasis of Lewis lung carcinoma cells to lung. Therefore, using beta-HIVS as a bioprobe, we investigated the molecular mechanism of shikonin's anti-angiogenic actions. beta-HIVS inhibited the phosphorylation and expression of VEGFR2 and Tie2 without affecting VEGFR1 and fibroblast growth factor receptor 1 levels. beta-HIVS suppressed the phosphorylation but not the expression of extracellular signal-regulated kinase, and an Sp1-dependent transactivation of the VEGFR2 and Tie2 promoters, thereby suppressing the proliferation of vascular endothelial and progenitor cells. This was mimicked by an Sp1 inhibitor mithramycin A and partially rescued by Sp1 overexpression. These results implicate potential use of shikonin and beta-HIVS as leading compounds for clinical application in the future by virtue of their unique properties including: (i) inhibition of VEGFR2 and Tie2 phosphorylation in an adenosine triphosphate-non-competitive manner; (ii) simultaneous inhibition of the phosphorylation and expression of VEGFR2 and Tie2; and (iii) bifunctional inhibition of the growth in endothelial cells and vascular remodeling.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Medicamentos Herbarios Chinos/farmacología , Naftoquinonas/farmacología , Neovascularización Patológica/prevención & control , Animales , Apoptosis , Western Blotting , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Adhesión Celular , Movimiento Celular , Proliferación Celular , Células Cultivadas , Pollos , Membrana Corioalantoides/metabolismo , Membrana Corioalantoides/patología , Ensayo de Cambio de Movilidad Electroforética , Femenino , Técnicas para Inmunoenzimas , Inmunoprecipitación , Luciferasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosforilación , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Transfección , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
In addition to humoral angiogenic factors, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), integrin-mediated adhesion of vascular endothelial cells to the extracellular matrix plays an important role in neovascularization. We recently found that TNIIIA2, a peptide derived from tenascin-C, induces functional activation of beta1 integrins. Here we investigated the effect of TNIIIA2 on vascular endothelial cell migration and proliferation, key processes for angiogenesis. TNIIIA2 was shown to activate beta1-integrins on human dermal microvascular endothelial cells (HDMEC). HDMEC adhered to fibronectin mainly via integrin alpha5beta1 and their haptotactic migration on that substrate was inhibited by TNIIIA2, in concomitant with a marked inhibition of Rac activation. TNIIIA2-treatment unaffected autophosphorylation of focal adhesion kinase (FAK), but induced its physical association with phospho-paxillin (Tyr118), suggesting the FAK/paxillin-dependent negative regulation of Rac activation. HDMEC proliferation on the fibronectin substrate was also inhibited by TNIIIA2-treatment, and this was accompanied either by an increase in the population of G 0/G1 cells and, conversely, a decrease in the population of S and G2/M cells or by dephosphorylation/inactivation of MAP-kinase (ERK1/2). Inhibited HDMEC migration and proliferation were both restored by pretreating the cells with a fibronectin peptide, FNIII14, which is capable of inactivating beta1-integrins. The chorioallantoic membrane assay demonstrated an antiangiogenic effect of TNIIIA2 in vivo. Thus, TNIIIA2 appears to negatively regulate angiogenesis by inhibiting migration and proliferation of endothelial cells. The ability to activate beta1-integrins may be responsible for the antiangiogenic effect of TNIIIA2, although it cannot be excluded the possibility that an additional mechanism(s) may play a role.
Asunto(s)
Inhibidores de la Angiogénesis , Integrina beta1/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Tenascina/farmacología , Animales , Biotransformación/efectos de los fármacos , Western Blotting , Ciclo Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Embrión de Pollo , Membrana Corioalantoides/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Citometría de Flujo , Humanos , Inmunoprecipitación , Proteínas Quinasas Activadas por Mitógenos/biosíntesis , Heridas y Lesiones/patología , Proteínas de Unión al GTP rac/biosíntesis , Proteínas de Unión al GTP rho/metabolismoRESUMEN
We designed chiral 2-nitroimidazole derivatives containing a 2-aminomethylene-4-cyclopentene-1,3-dione moiety as antiangiogenic hypoxic cell radiosensitizers. Based on results of molecular orbital calculations, the 2-aminomethylene-4-cyclopentene-1,3-dione moiety is expected to show high electrophilicity comparable to that of the 2-methylene-4-cyclopentene-1,3-dione moiety included in TX-1123 and tyrphostin AG17. We evaluated the antiangiogenic and radiosensitizing effects of the new compounds, along with other biological properties including their activities as hypoxic cytotoxicities and protein tyrosine kinase (PTK) inhibitory activities. Among the compounds tested, 5 (TX-2036) proved to be the strongest antiangiogenic hypoxic cell radiosensitizer. All the other chiral 2-nitroimidazole derivatives having 2-aminomethylene-4-cyclopentene-1,3-dione moiety tested were also antiangiogenic hypoxic cell radiosensitizers. The PTK inhibitory activity of 5 (TX-2036) showed this to be a promising and potent EGFR kinase inhibitor, having an IC(50) value of lower than 2microM. This compound also was an Flt-1 kinase inhibitor having an IC(50) value of lower than 20microM. Our results show that these chiral 2-nitroimidazole derivatives that contain the 2-aminomethylene-4-cyclopentene-1,3-dione moiety as a potent antiangiogenic pharmacophoric descriptor are promising lead candidates for the development of antiangiogenic hypoxic cell radiosensitizers.
Asunto(s)
Inhibidores de la Angiogénesis/síntesis química , Hipoxia de la Célula/efectos de los fármacos , Ciclopentanos/síntesis química , Diseño de Fármacos , Nitroimidazoles/química , Fármacos Sensibilizantes a Radiaciones/síntesis química , Inhibidores de la Angiogénesis/toxicidad , Animales , Antineoplásicos/síntesis química , Antineoplásicos/toxicidad , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Embrión de Pollo , Ciclopentanos/toxicidad , Ratones , Modelos Moleculares , Nitroimidazoles/toxicidad , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/toxicidad , Fármacos Sensibilizantes a Radiaciones/toxicidad , Ratas , EstereoisomerismoRESUMEN
BACKGROUND: Sangivamycin, an antibiotic with anti-tumor and anti-herpes virus activities by inhibiting both DNA/RNA synthesis and protein kinase C activity, was reported to suppress selectively DNA synthesis and growth of human umbilical vein endothelial cells and their tube formation in vitro. Here, to address the potential clinical use of sangivamycin in future, we investigated its anti-angiogenic effect in in vivo chicken chorioallantoic membrane (CAM) and mouse dorsal air sac (DAS) assays, and investigated underlying mechanism. METHODS: The effect of sangivamycin on blood vessel formation in CAM was observed under the microscope after treating for two days. For DAS assays, chambers fulfilled with tumor cells were implanted beneath mouse dorsal skin. After the mice were administered with sangivamycin, tumor-induced angiogenesis was observed under the microscope. The effect of sangivamycin on ATP synthesis on the endothelial cell surface was assayed by measuring ATP production with bioluminescence assay. RESULTS: Sangivamycin suppressed angiogenesis within CAM down to 94-71%, which was partially blocked by simultaneous addition of a 40-fold excess of adenosine. Sangivamycin also inhibited tumor-angiogenesis in the DAS assay by 61%, and suppressed ATP production on the endothelial cell surface by 75%. CONCLUSION: Sangivamycin inhibits the in vivo angiogenesis within CAM and tumor-induced angiogenesis within mouse dorsal skin, at least in part via inhibiting endothelial cell surface ATP metabolism in addition to inhibition of DNA/RNA synthesis and/or protein kinase C activity, suggesting a potential clinical use of sangivamycin as a novel anti-cancer reagent capable of targeting not only cancer cells but also endothelial cells.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antibióticos Antineoplásicos/farmacología , Membrana Corioalantoides/irrigación sanguínea , Membrana Corioalantoides/efectos de los fármacos , Endotelio/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Nucleósidos de Pirimidina/farmacología , Sacos Aéreos , Animales , Línea Celular , Pollos , Femenino , Ratones , Ratones Endogámicos ICR , Neovascularización Patológica/tratamiento farmacológico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tumor-related angiogenesis is likely to be a potential target for the treatment of cancer. One key to develop this angiostatic strategy would be to find useful angiogenesis inhibitors. Here we report the effects of radicicol, a microbial angiogenesis inhibitor that we previously identified using the chorioallantoic membrane assay, and its novel analog, 14,16-dipalmitoyl-radicicol, on tumor angiogenesis and growth. As expected for agents containing a penolic hydroxyl group, systemic administration of radicicol had little or no effect on neovascularization triggered by a M5076 mouse tumor cell line or a RMT-1 rat mammary carcinoma cell line established from autochthonous rat mammary tumors induced by 7,12-dimethylbenz[a]anthracene in a mouse dorsal air sac assay system. The agent did not show growth-inhibitory activity against either transplantable M5076 tumors or autochthonous 7,12-dimethylbenz[a]anthracene-induced rat mammary tumors. In contrast, 14,16-dipalmitoyl-radicicol potently suppressed tumor angiogenesis and growth in these experimental models. Furthermore, the analog significantly prolonged the survival rate of M5076-implanted mice. Although not stronger than radicicol, it dose-dependently inhibited embryonic angiogenesis in the chorioallantoic membrane assay, the dose required for half-maximal inhibition (ID(50)) value being 23 microg (27 nmol) per egg, and showed concentration-dependent antiproliferative activity against microvascular endothelial cells in vitro. These data suggest that 14,16-dipalmitoyl-radicicol is a promising antitumor agent with antiangiogenic activity.
Asunto(s)
Antineoplásicos/uso terapéutico , Ácidos Grasos Monoinsaturados/química , Macrólidos/química , Macrólidos/uso terapéutico , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Neovascularización Patológica/tratamiento farmacológico , Sacos Aéreos/efectos de los fármacos , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Membrana Corioalantoides/irrigación sanguínea , Membrana Corioalantoides/efectos de los fármacos , Membrana Corioalantoides/patología , Femenino , Macrólidos/farmacología , Ratones , Estructura Molecular , Trasplante de Neoplasias , Neoplasias/irrigación sanguínea , Neovascularización Patológica/patología , Ratas , Tasa de SupervivenciaRESUMEN
IL-27 is a novel IL-6/IL-12 family cytokine playing an important role in the early regulation of Th1 responses. We have recently demonstrated that IL-27 has potent antitumor activity, which is mainly mediated through CD8(+) T cells, against highly immunogenic murine colon carcinoma. In this study, we further evaluated the antitumor and antiangiogenic activities of IL-27, using poorly immunogenic murine melanoma B16F10 tumors, which were engineered to overexpress single-chain IL-27 (B16F10 + IL-27). B16F10 + IL-27 cells exerted antitumor activity against not only s.c. tumor but also experimental pulmonary metastasis. Similar antitumor and antimetastatic activities of IL-27 were also observed in IFN-gamma knockout mice. In NOD-SCID mice, these activities were decreased, but were still fairly well-retained, suggesting that different mechanisms other than the immune response are also involved in the exertion of these activities. Immunohistochemical analyses with Abs against vascular endothelial growth factor and CD31 revealed that B16F10 + IL-27 cells markedly suppressed tumor-induced neovascularization in lung metastases. Moreover, B16F10 + IL-27 cells clearly inhibited angiogenesis by dorsal air sac method, and IL-27 exhibited dose-dependent inhibition of angiogenesis on chick embryo chorioallantoic membrane. IL-27 was revealed to directly act on HUVECs and induce production of the antiangiogenic chemokines, IFN-gamma-inducible protein (IP-10) and monokine induced by IFN-gamma. Finally, augmented mRNA expression of IP-10 and monokine induced by IFN-gamma was detected at the s.c. B16F10 + IL-27 tumor site, and antitumor activity of IL-27 was partially inhibited by the administration of anti-IP-10. These results suggest that IL-27 possesses potent antiangiogenic activity, which plays an important role in its antitumor and antimetastatic activities.
Asunto(s)
Inhibidores de la Angiogénesis/fisiología , Antineoplásicos/farmacología , Interleucinas/fisiología , Inhibidores de la Angiogénesis/administración & dosificación , Animales , Antineoplásicos/administración & dosificación , Línea Celular , Embrión de Pollo , Inhibidores de Crecimiento/fisiología , Humanos , Inyecciones Subcutáneas , Interferón gamma/deficiencia , Interferón gamma/genética , Interleucinas/farmacología , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Trasplante de Neoplasias , Neovascularización Patológica/prevención & control , Proteínas Recombinantes/farmacologíaRESUMEN
(R)- and (S)-Epichlorohydrins were used to prepare the enantiomers of sterically diverse haloacetylcarbamoyl-2-nitroimidazoles that function as hypoxic cell radiosensitizers. The synthetic design allowed for introduction of a side chain of varying bulk that permitted an examination of the steric effects on enantio-discrimination in biological assay systems. The single stereocenter also connected the two pharmacophores--a 2-nitroimidazole moiety critical to hypoxic cell radiosensitization, and a haloacetylcarbamoyl group to function as an anti-angiogenesis pharmacophore. In the chick embryo chorioallantoic membrane (CAM) assay, the R-enantiomers possessing the bulky p-tert-butylphenyl group showed higher anti-angiogenic activity than the corresponding S-enantiomers, while there were no differences in the activity between the enantiomers containing the less bulky methyl and tert-butyl groups. Among the compounds we report, R-p-tert-butylphenyl-bromoacetylcarbamoyl-2-nitroimidazole, TX-1898, was found to be the most promising candidate for further development of as anti-angiogenic hypoxic cell radiosensitizer.
Asunto(s)
Inhibidores de la Angiogénesis/síntesis química , Nitroimidazoles/síntesis química , Fármacos Sensibilizantes a Radiaciones/síntesis química , Inhibidores de la Angiogénesis/farmacología , Animales , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/fisiología , Embrión de Pollo , Nitroimidazoles/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , EstereoisomerismoRESUMEN
Recent studies have demonstrated that bovine lactoferrin (bLF) suppresses tumor growth and metastasis in the mouse and rat and moreover may inhibit angiogenesis. To determine whether angiogenesis inhibition might contribute to antitumor activity, we examined the influence of bLF on tumor-induced angiogenesis and endothelial cell functions as well as angiogenesis-related cytokine production. Bovine LF exhibited dose-dependent inhibition of angiogenesis on 4-6-day-old chick embryo chorioallantoic membranes (CAMs) that lack a mature immune response. This inhibition was reversed when bLF was simultaneously treated with basic fibroblast growth factor (bFGF). It also inhibited in vitro formation of tube-like structures of mouse endothelial KOP2.16 cells. Moreover, it potently suppressed bFGF- or VEGF-induced proliferation of mouse endothelial KOP2.16 cells, but not of mouse fibroblast A31 cells and Lewis lung carcinoma (3LL) cells. In mice, both orally and intraperitoneally administered bLF significantly and dose-dependently suppressed 3LL cell-induced angiogenesis in a dorsal air sac assay. As orally administered bLF was reported to exhibit antitumor activity through production of interferon (IFN)-gamma and interleukin (IL)-18 in intestinal mucosa (Kuhara T et al., Nutr Cancer 2000;38:192-9), production of these cytokines in mouse serum and peritoneal macrophages by bLF was examined. IFN-gamma was not detected in serum by bLF administration. However, bLF markedly elevated IL-18 concentration in serum by oral administration, but not by intraperitoneal administration. It also induced IL-18 in peritoneal macrophages in vitro. These results suggest that bLF participates as a regulator of angiogenesis, possibly explained by blocking endothelial function and inducing IL-18 production. Antitumor activity of bLF may thus be partly mediated by angiogenesis inhibition.
Asunto(s)
Lactoferrina/farmacología , Neovascularización Patológica , Administración Oral , Animales , Bovinos , Embrión de Pollo , Citocinas/farmacología , Relación Dosis-Respuesta a Droga , Células Endoteliales/fisiología , Femenino , Infusiones Parenterales , Interleucina-18/farmacología , Lactoferrina/administración & dosificación , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Neoplasias/irrigación sanguíneaRESUMEN
We review in this report our strategy and tactics for the design of 2-hydroxyarylidene-4-cyclopentene-1,3-diones as protein tyrosine kinase (PTK) inhibitors having low mitochondrial toxicities and/or hypoxia-targeting function. We based our synthetic design on an innovative pharmacophore, 2-methylene-4-cyclopentene-1,3-dione. We first showed the effectiveness of this pharmacophore in the development of 2-methylene-4-cyclopentene-1,3-dione as PTK inhibitor that have lower mitochondrial toxicity than the potent PTK inhibitor tyrphostin AG17. Our results show that the cyclopentenedione-derived TX-1123 is a more potent antitumor tyrphostin and also shows lower mitochondrial toxicity than the malononitrile-derived AG17. The O-methylation product of TX-1123 (TX-1925) retained its tyrphostin-like properties, including mitochondrial toxicity and antitumor activities. However, the methylation product of AG17 (TX-1927) retained its tyrphostin-like antitumor activities, but lost its mitochondrial toxicity. Our comprehensive evaluation of these agents with respect to PTK inhibition, mitochondrial inhibition, antitumor activity, and hepatotoxicity demonstrates that PTK inhibitors TX-1123 and TX-1925 are more promising candidates for antitumor agents than tyrphostin AG17. Secondly, as a further investigation of the promising power of this 4-cyclopentene-1,3-dione as an innovative pharmacophore, we discuss our strategy of development of hypoxia-targeting PTK inhibitor TX-1123 analogues, 2-nitroimidazole-aminomethylenecyclopentenediones, such as TX-2036, for cancer treatment, especially for pancreatic cancers, which have a high level of hypoxia.
Asunto(s)
Hipoxia de la Célula/efectos de los fármacos , Ciclopentanos/química , Ciclopentanos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Compuestos de Bencilideno/química , Compuestos de Bencilideno/farmacología , Compuestos de Bencilideno/toxicidad , Hipoxia de la Célula/fisiología , Ciclopentanos/toxicidad , Diseño de Fármacos , Inhibidores Enzimáticos/toxicidad , Humanos , Mitocondrias/efectos de los fármacos , Modelos Moleculares , Nitrilos , Relación Estructura-Actividad , Tirfostinos/química , Tirfostinos/farmacología , Tirfostinos/toxicidadRESUMEN
We have previously demonstrated that oral administration of bovine lactoferrin (bLF) markedly inhibits lung metastatic colony formation, and that this inhibition was possibly due to the activation of T and NK cells. Furthermore, we found that interleukin-18 (IL-18) is induced in epithelial cells of the small intestine by bLF. The present study was undertaken to confirm cytokine production in response to bLF and to assess the underlying mechanisms. Markedly elevated IL-18 levels were found in the small intestine 1-3 h after a single administration of bLF, its pepsin hydrolysate (bLFH), or bTF. Importantly, while IL-18 was significantly increased after a regimen of seven daily administrations of bLF or bLFH, administration of bTF over the course of seven days had little or no effect. In addition to IL-18, a significant increase in caspase-1 activity and interferon-gamma (IFN-gamma) was found in the small intestine after administration of bLF. Similarly, in peritoneal macrophages, bLF markedly enhanced caspase-1 activity and IL-18 levels. Finally, a caspase-1 inhibitor significantly decreased bLF mediated induction of IL-18 in vitro. (bTF had no effect on either caspase-1 or IFN-gamma or on IL-18 in vitro.) These results demonstrate the possibility that elevation of caspase-1 activity by bLF and its hydrolysate may be important for production of mature IL-18 in vivo, and thus in potentiating the killing activity of T and NK cells against tumor cells.
Asunto(s)
Caspasa 1/metabolismo , Interleucina-18/metabolismo , Mucosa Intestinal/efectos de los fármacos , Lactoferrina/administración & dosificación , Administración Oral , Clorometilcetonas de Aminoácidos/farmacología , Animales , Caspasa 1/efectos de los fármacos , Inhibidores de Caspasas , Bovinos , Células Cultivadas , Inhibidores de Cisteína Proteinasa/farmacología , Citocinas/metabolismo , Hidrólisis , Interferón gamma/sangre , Interferón gamma/metabolismo , Interleucina-18/sangre , Mucosa Intestinal/metabolismo , Intestinos/efectos de los fármacos , Lactoferrina/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Neoplasias/metabolismo , Neoplasias/patología , Pepsina A/metabolismoRESUMEN
Endothelial permeability is increased by vascular endothelial cell growth factor and decreased by antioxidants. Whether or not l-ascorbic acid (Asc), which decreases endothelial permeability by stimulating the endothelial barrier function, is anti-angiogenic (angiostatic) remains unknown. We examined the role of Asc on angiogenesis using two assay systems. At first, the potential role of Asc on four steps of angiogenesis was investigated in cultured bovine microvascular endothelial cells. Asc inhibited the formation of vessel-like tubular structures of endothelial cells cultured on Matrigel; however, it did not decrease the activity of plasminogen activator (PA), which creates the space into which vascular vessels extend. Furthermore, even at high concentrations, Asc did not inhibit either the proliferation or migration of endothelial cell cultures. Secondly, whether Asc inhibited in vivo angiogenesis or not was studied on chick chorioallantoic membrane (CAM) during the 4-6 days of embryogenesis when neovascularization is rapid. It also revealed that angiogenesis was dose-dependently inhibited by Asc from 0.5 micro mol/CAM with half-maximal inhibition at 2.5 micro mol/CAM. Because it was previously reported that the endothelial barrier function decreases permeability via the stimulation of collagen synthesis induced by Asc, we treated CAM with the inhibitor of collagen synthesis, l-azetidine 2-carboxylic acid (AzC). This compound partially attenuated the angiostatic function of Asc on CAM. To understand the involvement of an antioxidant activity in the angiostatic function of Asc, we further examined the effect of glutathione (GSH), which is an endogenous antioxidant, on angiogenesis in CAM and endothelial cells. GSH inhibited CAM angiogenesis, as well as the formation of vessel-like tubular structures of endothelial cell cultures on Matrigel. Both Asc and GSH inhibited hydrogen peroxide (H(2)O(2)) induced tubular morphogenesis. These findings suggest that Asc affects angiogenesis through both its antioxidant properties and the stimulation of collagen synthesis. As the angiostatic activity of Asc may be one of the many effects involved in host resistance to the growth or invasiveness of solid cancer, it may be useful as a supplementary therapy in various angiogenic diseases.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Ácido Ascórbico/farmacología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Animales , Antioxidantes/farmacología , Ácido Azetidinocarboxílico/farmacología , Capilares/citología , Bovinos , Línea Celular , Células Cultivadas , Embrión de Pollo , Colágeno/biosíntesis , Colágeno/química , Colágeno/efectos de los fármacos , Colágeno/metabolismo , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Glutatión/farmacología , Peróxido de Hidrógeno/farmacología , Laminina/química , Laminina/metabolismo , Oxidantes/farmacología , Activadores Plasminogénicos/análisis , Proteoglicanos/química , Proteoglicanos/metabolismoRESUMEN
For cancer therapy, hypoxia represents an important tumor specific target. Therefore we designed and synthesized antiangiogenic hypoxic cytotoxins as 'hypoxia modifiers'. They can be activated bioreductively in hypoxic cells to kill the oxygen-deficient tumor cells selectively and prevent their re-growth. The aromatic heterocycle di-N-oxides, tirapazamine (TPZ), TX-1102, and TX-402 inhibited growth of EMT6/KU cells, SAS/neo cells, and SAS/Trp248 cells (mutant p53 gene transformant) under hypoxic condition. They also induced apoptosis selectively at a dose of 10 microM each under hypoxic condition for 5 h. Their hypoxic cytotoxicities and apoptosis inducing activities were p53-independent because the activities in SAS/neo cells were almost similar to that in SAS/Trp248 cells. In angiogenesis inhibition assay using chick embryo chorioallantoic membrane (CAM), TPZ, TX-1102, TX-402 and TX-1033 showed 40, 25, 60 and 60% inhibition of angiogenesis each at a dose of 10 microg/CAM. On the other hand, the nitrosopyrimidine, TX-1041 had neither antiangiogenic activity nor cytotoxicity. Therefore the di-N-oxide group is thought to be required for the biological activities. TX-1102 was a potent antiangiogenic hypoxic cytotoxin inducing apoptosis p53-independently.
Asunto(s)
Inhibidores de la Angiogénesis/síntesis química , Inhibidores de la Angiogénesis/farmacología , Citotoxinas/síntesis química , Diseño de Fármacos , Neovascularización Patológica/tratamiento farmacológico , Triazinas/síntesis química , Aerobiosis , Inhibidores de la Angiogénesis/química , Animales , Apoptosis/efectos de los fármacos , Hipoxia de la Célula/efectos de los fármacos , Embrión de Pollo , Citotoxinas/química , Citotoxinas/farmacología , Fragmentación del ADN/efectos de los fármacos , Humanos , Triazinas/química , Triazinas/farmacología , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
We designed and synthesized the antiangiogenic arginine-rich dendrimers, TX-1943 and TX-1944, which mimic the surface structure of endostatin. TX-1944 containing 16 arginine residues is more similar in surface structure to endostatin than TX-1943 with eight arginine residues, and has stronger in vivo antiangiogenic activity at 10 microg/CAM in the chicken embryo chorioallantoic membrane (CAM) assay. TX-1944 also has stronger activity to bind heparin and to inhibit the growth of rat lung endothelial (RLE) cells than TX-1943.