RESUMEN
Galectin-1 (gal-1), which binds ß-galactoside groups on various cell surface receptors, is crucial to cell adhesion and migration, and is found to be elevated in several cancers. Previously, we reported on 6DBF7, a dibenzofuran (DBF)-based peptidomimetic of the gal-1 antagonist anginex. In the present study, we used a structure-based approach to optimize 6DBF7. Initial NMR studies showed that 6DBF7 binds to gal-1 on one side of the ß-sandwich away from the lectin's carbohydrate binding site. Although an alanine scan of 6DBF7 showed that the two cationic groups (lysines) in the partial peptide are crucial to its angiostatic activity, it is the hydrophobic face of the amphipath that appears to interact directly with the surface of gal-1. Based on this structural information, we designed and tested additional DBF analogs. In particular, substitution of the C-terminal Asp for alanine and branched alkyl side chains (Val, Leu, Ile) for linear ones (Nle, Nva) rendered the greatest improvements in activity. Flow cytometry with gal-1(-/-) splenocytes showed that 6DBF7 and two of its more potent analogs (DB16 and DB21) can fully inhibit fluorescein isothiocyanate-gal-1 binding. Moreover, heteronuclear single-quantum coherence NMR titrations showed that the presence of DB16 decreases gal-1 affinity for lactose, indicating that the peptidomimetic targets gal-1 as a noncompetitive, allosteric inhibitor of glycan binding. Using tumor mouse models (B16F10 melanoma, LS174 lung, and MA148 ovarian), we found that DB21 inhibits tumor angiogenesis and tumor growth significantly better than 6DBF7, DB16, or anginex. DB21 is currently being developed further and holds promise for the management of human cancer in the clinic.
Asunto(s)
Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/farmacología , Benzofuranos/química , Benzofuranos/farmacología , Galectina 1/antagonistas & inhibidores , Alanina/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Línea Celular Tumoral , Femenino , Galectina 1/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Lactosa/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Datos de Secuencia Molecular , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Peptidomiméticos/química , Peptidomiméticos/farmacología , Unión Proteica , Relación Estructura-ActividadRESUMEN
The purpose of this study was to delineate the mechanisms by which stromal components of cancer may induce tumour thermotolerance and exploit alterations in stromal and tumour physiology to enhance radiation therapy. The vascular thermoresponse was monitored by daily one-hour 41.5°C heatings in two murine solid tumour models, SCK murine mammary carcinoma and B16F10 melanoma. A transient increase was seen in overall tumour oxygenation for 2-3 days, followed by a progressive decline in tumour pO(2) upon continued daily heatings. Vascular thermotolerance was further studied by treating tumours with different heating strategies, i.e. (1) a single 60 min 41.5°C treatment; (2) two consecutive daily treatments of 41.5°C for 60 min; (3) a single 60 min 43°C treatment or (4) two days of 41.5°C for 60 min followed by treatment with 43°C for 60 min on the third day. Pre-heating tumours with mild temperature hyperthermia induced vascular thermotolerance, which was accompanied by evidence of vessel normalisation, i.e. a decrease in microvessel density and an increase in pericyte coverage. Rational scheduling of fractionated radiation during heat-induced increases in tumour oxygen levels rendered a significantly greater, synergistic, tumour growth inhibition. In vitro clonogenic survival responses of the individual cell types associated (endothelial cells, fibroblasts, pericytes and tumour cells) indicated only a direct cellular thermotolerance in endothelial cells. Overall, this suggests that tumour thermotolerance is a physiological phenomenon mediated through improvement of functional vasculature.
Asunto(s)
Hipertermia Inducida , Neoplasias/irrigación sanguínea , Animales , Terapia Combinada , Femenino , Masculino , Neoplasias Mamarias Experimentales/irrigación sanguínea , Neoplasias Mamarias Experimentales/fisiopatología , Neoplasias Mamarias Experimentales/radioterapia , Neoplasias Mamarias Experimentales/terapia , Ratones , Neoplasias/fisiopatología , Neoplasias/radioterapia , Neoplasias/terapia , Oxígeno/metabolismo , Presión ParcialRESUMEN
Targeted delivery of therapeutic drugs promises to become the norm to treat cancer. Here, we conjugated the cytotoxic agent 6-hydroxypropylacylfulvene (HPAF) to anginex, a peptide that targets galectin-1, which is highly expressed in endothelial cells of tumor vessels. In a human ovarian cancer model in mice, the conjugate inhibited tumor growth better than equivalent doses of either compound alone. Immunofluorescence on tumor tissue demonstrated that the conjugate, like parent anginex, selectively targeted tumor vasculature and inhibited tumor angiogenesis. Increased activity from the conjugate further suggests that HPAF retains at least some of its normal cytotoxic activity when linked to anginex. More importantly perhaps is the observation that the conjugate abrogates apparent systemic toxicity from treatment with HPAF. This work contributes to the development of tumor vascular targeting agents against cancer in the clinic.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/farmacología , Galectina 1/antagonistas & inhibidores , Neoplasias/irrigación sanguínea , Neoplasias/patología , Neovascularización Patológica/tratamiento farmacológico , Proteínas/química , Inhibidores de la Angiogénesis/uso terapéutico , Antineoplásicos/uso terapéutico , División Celular/efectos de los fármacos , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Galectina 1/metabolismo , Humanos , Neoplasias/tratamiento farmacológico , Péptidos , Proteínas/farmacología , Proteínas/uso terapéutico , Sesquiterpenos/química , Sesquiterpenos/farmacología , Sesquiterpenos/uso terapéutico , Compuestos de Espiro/química , Compuestos de Espiro/farmacología , Compuestos de Espiro/uso terapéutico , Células Tumorales Cultivadas , Venas Umbilicales/citologíaRESUMEN
PURPOSE: The current work describes the synergistic enhancement of hyperthermic cancer therapy by selective thermal sensitization and induction of vascular injury at the tumor site. The specificity of this response was mediated by CYT-6091: a pegylated colloidal gold-based nanotherapeutic designed to selectively deliver an inflammatory cytokine, tumor necrosis factor alpha (TNF), to solid tumors. MATERIALS AND METHODS: FSaII murine fibrosarcoma-bearing C3H mice received an intravenous injection of either soluble TNF or CYT-6091 (50-250 microg/kg TNF). Four hours later the tumors were exposed to localized heating (42.5 or 43.5 degrees C, 60 min). Tumor responses were assessed by growth delay and/or perfusion. RESULTS: Both soluble TNF and CYT-6091 reduced tumor perfusion by 80% of control (no treatment), 4 hours post administration. However, soluble TNF was toxic to the tumor burdened mice and resulted in 40% mortality alone and 100% mortality when combined with hyperthermia. Conversely, no toxicities were noted with CYT-6091 alone or when combined with hyperthermia. Additionally, CYT-6091 combined with heat yielded significant tumor regression in vivo as compared to heat or CYT-6091 alone as demonstrated by tumor growth delay. Pretreatment with soluble TNF or CYT-6091 followed by heating reduced in vitro tumor and endothelial cell survival by 40-50% (TNF) and 70-75% (CYT-6091) of the control cell (i.e. tumor and endothelial) values, respectively. CONCLUSIONS: CYT-6091, by selectively delivering TNF to solid tumors, improves the safety of TNF treatment. In addition, the targeted delivery of TNF augments cancer thermal therapy efficacy possibly by inducing a tumor-localized inflammatory response.
Asunto(s)
Portadores de Fármacos/uso terapéutico , Fibrosarcoma/terapia , Hipertermia Inducida/métodos , Nanoestructuras/uso terapéutico , Factor de Necrosis Tumoral alfa/uso terapéutico , Animales , Línea Celular Tumoral , Terapia Combinada , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Femenino , Oro Coloide , Humanos , Ratones , PolietilenglicolesRESUMEN
We found that beta-lapachone (beta-lap), a novel bioreductive drug, caused rapid apoptosis and clonogenic cell death in A549 human lung epithelial cancer cells in vitro in a dose-dependent manner. The clonogenic cell death caused by beta-lap could be significantly inhibited by dicoumarol, an inhibitor of NAD(P)H:quinone oxido-reductase (NQO1), and also by siRNA for NQO1, demonstrating that NQO1-induced bioreduction of beta-lap is an essential step in beta-lap-induced cell death. Irradiation of A549 cells with 4 Gy caused a long-lasting upregulation of NQO1, thereby increasing NQO1-mediated beta-lap-induced cell deaths. Although the direct cause of beta-lap-induced apoptosis is not yet clear, beta-lap treatment reduced the expression of p53 and NF-kappaB, whereas it increased cytochrome C release, caspase-3 activity, and gammaH2AX foci formation. Importantly, beta-lap treatment immediately after irradiation enhanced radiation-induced cell death, indicating that beta-lap sensitizes cancer cells to radiation, in addition to directly killing some of the cells. The growth of A549 tumors induced in immunocompromised mice could be markedly suppressed by local radiation therapy when followed by beta-lap treatment. This is the first study to demonstrate that combined radiotherapy and beta-lap treatment can have a significant effect on human tumor xenografts.
Asunto(s)
NAD(P)H Deshidrogenasa (Quinona)/biosíntesis , NAD(P)H Deshidrogenasa (Quinona)/efectos de la radiación , Naftoquinonas/farmacología , Regulación hacia Arriba/efectos de la radiación , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Femenino , Humanos , Ratones , Ratones Desnudos , NAD(P)H Deshidrogenasa (Quinona)/fisiología , Radiación Ionizante , Regulación hacia Arriba/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
PURPOSE: To test whether a direct antiangiogenic peptide (anginex) and a vascular endothelial growth factor antibody (bevacizumab, Avastin) can transiently normalize vasculature within tumors to improve oxygen delivery, alleviate hypoxia, and increase the effect of radiation therapy. EXPERIMENTAL DESIGN: Tumor oxygenation levels, microvessel density and pericyte coverage were monitored in three different solid tumor models (xenograft human ovarian carcinoma MA148, murine melanoma B16F10, and murine breast carcinoma SCK) in mice. Multiple treatment schedules were tested in these models to assess the influence on the effect of radiation therapy. RESULTS: In all three tumor models, we found that tumor oxygenation levels, monitored daily in real time, were increased during the first 4 days of treatment with both anginex and bevacizumab. From treatment day 5 onward, tumor oxygenation in treated mice decreased significantly to below that in control mice. This "tumor oxygenation window" occurred in all three tumor models varying in origin and growth rate. Moreover, during the treatment period, tumor microvessel density decreased and pericyte coverage of vessels increased, supporting the idea of vessel normalization. We also found that the transient modulation of tumor physiology caused by either antiangiogenic therapy improved the effect of radiation treatment. Tumor growth delay was enhanced when single dose or fractionated radiotherapy was initiated within the tumor oxygenation window as compared with other treatment schedules. CONCLUSIONS: The results are of immediate translational importance because the clinical benefits of bevacizumab therapy might be increased by more precise treatment scheduling to ensure radiation is given during periods of peak radiosensitivity. The oxygen elevation in tumors by non-growth factor-mediated peptide anginex suggests that vessel normalization might be a general phenomenon of agents directed at disrupting the tumor vasculature by a variety of mechanisms.