RESUMEN
BACKGROUND: Species selectivity of DMXAA (5,6-dimethylxanthenone-4-acetic acid, Vadimezan) for murine cells over human cells could explain in part the recent disappointing phase III trials clinical results when preclinical studies were so promising. To identify analogues with greater human clinical potential, we compared the activity of xanthenone-4-acetic acid (XAA) analogues in murine or human cellular models. METHODS: Analogues with a methyl group systematically substituted at different positions of the XAA backbone were evaluated for cytokine induction in cultured murine or human leukocytes; and for anti-vascular effects on endothelial cells on matrigel. In vivo antitumour activity and cytokine production by stromal or cancer cells was measured in human A375 and HCT116 xenografts. RESULTS: Mono-methyl XAA analogues with substitutions at the seventh and eighth positions were the most active in stimulating human leukocytes to produce IL-6 and IL-8; and for inhibition of tube formation by ECV304 human endothelial-like cells, while 5- and 6-substituted analogues were the most active in murine cell systems. CONCLUSION: Xanthenone-4-acetic acid analogues exhibit extreme species selectivity. Analogues that are the most active in human systems are inactive in murine models, highlighting the need for the use of appropriate in vivo animal models in selecting clinical candidates for this class of compounds.
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Antineoplásicos/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Xantenos/farmacología , Xantonas/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colágeno/metabolismo , Combinación de Medicamentos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Laminina/metabolismo , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Ratones , Ratones Desnudos , Proteoglicanos/metabolismo , Xantenos/químicaRESUMEN
BACKGROUND: The non-malignant cells of the tumour stroma have a critical role in tumour biology. Studies dissecting the interplay between cancer cells and stromal cells are required to further our understanding of tumour progression and methods of intervention. For proof-of-principle of a multi-modal approach to dissect the differential effects of treatment on cancer cells and stromal cells, we analysed the effects of the stromal-targeting agent 5,6-dimethylxanthenone-4-acetic acid on melanoma xenografts. METHODS: Flow cytometry and multi-colour immunofluorescence staining was used to analyse leukocyte numbers in xenografts. Murine-specific and human-specific multiplex cytokine panels were used to quantitate cytokines produced by stromal and melanoma cells, respectively. Human and mouse Affymetrix microarrays were used to separately identify melanoma cell-specific and stromal cell-specific gene expression. RESULTS: 5,6-Dimethylxanthenone-4-acetic acid activated pro-inflammatory signalling pathways and cytokine expression from both stromal and cancer cells, leading to neutrophil accumulation and haemorrhagic necrosis and a delay in tumour re-growth of 26 days in A375 melanoma xenografts. CONCLUSION: 5,6-Dimethylxanthenone-4-acetic acid and related analogues may potentially have utility in the treatment of melanoma. The experimental platform used allowed distinction between cancer cells and stromal cells and can be applied to investigate other tumour models and anti-cancer agents.
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Antineoplásicos/farmacología , Citocinas/metabolismo , Melanoma/patología , Xantonas/farmacología , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Citocinas/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Inactivación de Genes , Redes Reguladoras de Genes , Proteínas de Homeodominio/genética , Humanos , Leucocitos/efectos de los fármacos , Leucocitos/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Melanoma/metabolismo , Ratones , Ratones Noqueados , Ratones Desnudos , Análisis de Secuencia por Matrices de Oligonucleótidos , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , Transcripción Genética , Carga Tumoral/efectos de los fármacos , Regulación hacia Arriba , Xantonas/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Pre-eclampsia is characterized by systemic maternal endothelial dysfunction that precedes the onset of clinical symptoms. The cause of the dysfunction is not clear but the number and the nature of trophoblasts shed from the placenta may be altered in pre-eclamptic pregnancies. These dead trophoblasts become trapped in the pulmonary capillaries and may then be phagocytosed by endothelial cells. Phagocytosis of necrotic, but not apoptotic, trophoblasts results in endothelial cell activation. We have explored the hypothesis that activation can subsequently spread to other endothelial cells via soluble factors without the need for direct contact with shed trophoblasts. Conditioned medium from endothelial cells that had phagocytosed necrotic, but not apoptotic, trophoblasts was shown to activate fresh endothelial cells due, in large part, to IL-6 secreted into the conditioned medium. The amount of IL-6 secreted in response to phagocytosis of necrotic trophoblasts was similar to the levels of IL-6 found by others in the blood of pre-eclamptic women and was substantially more than the level of IL-6 which has been reported to induce symptoms of pre-eclampsia in pregnant rats. We demonstrated that phagocytosis of both a trophoblast cell line as well as trophoblasts shed from human placentae, had this effect on two different types of endothelial cells. The role of IL-6 in endothelial cell activation was confirmed using recombinant IL-6 and neutralizing antibodies against IL-6 and the IL-6 receptor. Thus, IL-6 secreted by pulmonary endothelial cells after they have phagocytosed necrotic trophoblasts that are trapped in the pulmonary capillaries could activate endothelial cells in other remote vascular beds, contributing to the systemic activation of the endothelium that is a hallmark of pre-eclampsia.
Asunto(s)
Endotelio Vascular/patología , Interleucina-6/fisiología , Fagocitosis , Preeclampsia/etiología , Trofoblastos/patología , Comunicación Celular , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Endotelio Vascular/metabolismo , Femenino , Humanos , Interleucina-6/biosíntesis , Interleucina-6/inmunología , Necrosis , Preeclampsia/metabolismo , Preeclampsia/patología , EmbarazoRESUMEN
Preeclampsia is a pregnancy-specific disease characterised by maternal hypertension that is preceded by endothelial cell activation and an inappropriate inflammatory response. The exact cause of preeclampsia is unclear but this disease is known to be induced by a placental factor and it is hypothesised that oxidative stress may also contribute to its pathogenesis. We have shown that dead trophoblasts shed from the placenta can be phagocytosed by endothelial cells and that phagocytosis of necrotic, but not apoptotic, trophoblasts leads to endothelial cells activation. Since phagocytosis may be accompanied by an oxidative burst which may lead to damage/activation of the phagocyte, in this study we have investigated whether the antioxidant vitamin C can protect endothelial cells that phagocytose necrotic trophoblasts from activation. We demonstrate that treatment of phagocytosing endothelial cells with vitamin C induced an increase in the phagocytosis of necrotic trophoblasts but that activation of the phagocytosing endothelial cells was prevented. Treatment of phagocytosing endothelial cells with vitamin C also prevented the increase in IL-6 secretion that normally accompanies phagocytosis of necrotic trophoblasts. Thus treatment of endothelial cells with vitamin C appears to modify both the phagocytosis of necrotic trophoblasts and the response of the endothelial cells to the necrotic trophoblastic material.
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Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Células Endoteliales/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Trofoblastos/efectos de los fármacos , Adulto , Línea Celular Tumoral , Coriocarcinoma/patología , Células Endoteliales/metabolismo , Femenino , Humanos , Interleucina-6/metabolismo , Necrosis , Fagocitosis/fisiología , Preeclampsia/metabolismo , Preeclampsia/fisiopatología , Embarazo , Trofoblastos/patología , Venas Umbilicales/citología , Adulto JovenRESUMEN
INTRODUCTION: Antiphospholipid antibodies (aPL) cause thrombotic disease and recurrent pregnancy loss. Despite their name it is now clear that the antigen for most antiphospholipid antibodies is the phospholipid-binding protein beta(2) glycoprotein I (beta(2)GPI). However, beta(2) glycoprotein I is only antigenic for antiphospholipid antibodies when the protein is immobilised on a suitable surface such as phosphatidyl serine. It has been suggested that antiphospholipid antibodies bind to beta(2) glycoprotein I on the surface of resting endothelial cells and this in turn leads to endothelial activation and the initiation of thrombosis. However, as phosphatidyl serine is absent from resting endothelial cell membranes, we questioned this hypothesis. MATERIALS AND METHODS: The ability of human antiphospholipid antibody-containing sera and monoclonal antiphospholipid antibodies to interact with endothelial cells was examined using cell-based ELISAs employing human umbilical vein endothelial cells (HUVECs) as the antigen. The expression of adhesion molecules in response to treatment with antiphospholipid antibodies was also measured by a cell-based ELISA. Activation of NF kappa beta was examined using electrophoretic mobility shift assays (EMSAs). RESULTS: Neither monoclonal antiphospholipid antibodies nor human sera containing antiphospholipid antibodies bound to resting endothelial cells. In contrast, one monoclonal antiphospholipid antibody did bind to both activated and apoptotic endothelial cells. CONCLUSIONS: Antiphospholipid antibodies do not bind to resting endothelial cells nor do antiphospholipid antibodies activate resting endothelial cells. Rather, an independent triggering event is required to activate endothelial cells and subsequently some antiphospholipid antibodies may then bind to the activated endothelial cells and initiate a thrombogenic process.
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Anticuerpos Antifosfolípidos/sangre , Anticuerpos Antifosfolípidos/inmunología , Síndrome Antifosfolípido/sangre , Síndrome Antifosfolípido/inmunología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/inmunología , Anticuerpos Antifosfolípidos/farmacología , Complejo Antígeno-Anticuerpo/inmunología , Células Cultivadas , Humanos , Activación Plaquetaria/efectos de los fármacos , Activación Plaquetaria/inmunología , Unión Proteica/inmunología , Venas UmbilicalesRESUMEN
5,6-Dimethylxanthenone-4-acetic acid (DMXAA) is currently undergoing clinical evaluation as an antivascular agent for the treatment of cancer. We have previously demonstrated that DMXAA induces apoptosis of vascular endothelial cells in murine tumour sections and in a breast carcinoma biopsy from one patient in a Phase I trial. We wished to determine the tissue selectivity of this effect and its relationship to induced blood flow changes. Mice with Colon 38 tumours were treated with DMXAA and tissues were examined for apoptosis by TdT-mediated dUTP nick-end labelling (TUNEL). Hoechst 33342 was used to stain functional vessels, with the loss of stained vessels used as a measure of tumour vascular collapse. Treatment with DMXAA at 25 mg kg(-1), its maximum tolerated dose (MTD), showed, after 3 h, a 12-fold increase in TUNEL staining of tumour vascular endothelial cells. In contrast, tissue from the heart, brain, liver and spleen showed no increase. Induction of apoptosis in tumour tissue was both dose-dependent, observable at doses as low as 5 mg kg(-1), and time-dependent. Apoptosis was significantly lower in Colon 38 tumours of mice, with a targeted disruption in the TNF gene (TNF(-/-)), or in the TNF receptor 1 gene (TNFR(-/-)), as compared with that in wild-type mice. Increasing the DMXAA dose to 50 mg kg(-1) in these knockout mice raised tumour apoptosis to a level comparable to that induced in wild-type mice given DMXAA at the MTD. For all the data, a significant correlation (r=0.94; P<0.001) was found between logarithmic percentage apoptosis induction and the logarithmic density of Hoechst-stained vessels. These results suggest that blood flow inhibition caused by DMXAA is tumour tissue-specific and is a consequence of induction of apoptosis in tumour vascular endothelial cells.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis , Neoplasias del Colon/irrigación sanguínea , Neoplasias del Colon/patología , Xantonas/farmacología , Animales , Neoplasias del Colon/veterinaria , Células Endoteliales/fisiología , Etiquetado Corte-Fin in Situ , Ratones , Ratones Noqueados , Neoplasias Experimentales , Flujo Sanguíneo Regional , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a new anticancer agent developed in this centre, has an antivascular action and causes regression of transplantable murine tumours that is mediated partially by the intratumoral production of tumour necrosis factor (TNF). DMXAA activates the nuclear factor-kappaB (NF-kappaB) transcription factor, which is involved in TNF synthesis and has also been suggested to mediate resistance to TNF. We wished to determine whether tumour cell NF-kappaB activation modulated the in vitro and in vivo effects of DMXAA. We compared the response of the 70Z/3 pre-B lymphoma cell line with that of its mutant 1.3E2 sub-line, which has a defective gamma-subunit of IKK, the kinase that phosphorylates IkappaB leading to NF-kappaB activation. As shown by electrophoretic mobility shift assays (EMSAs), DMXAA induced in vitro translocation of NF-kappaB (p50 and p65 subunits) into the nucleus of 70Z/3 cells, but not of 1.3E2 cells. However, when the cell lines were then grown as subcutaneous tumours in mice and treated with DMXAA (25 mg/kg), activation of NF-kappaB was found in nuclear extracts prepared from both 70/Z3 and 1.3E2 tumours, as well as from Colon 38 tumours that were used for comparison. This suggests that DMXAA induces NF-kappaB responses in host components of the tumour. Tumours grown from both 70Z/3 and 1.3E2 cells were found to regress completely following DMXAA treatment. Thus, the antitumour action of DMXAA appears to be independent of the ability of the target tumour cell population to induce NF-kappaB expression. Moreover, activation of NF-kappaB in the tumour cell did not confer resistance to DMXAA-induced therapy.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Antineoplásicos/uso terapéutico , FN-kappa B/metabolismo , Xantenos/uso terapéutico , Xantonas , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/patología , Inhibidores de la Angiogénesis/farmacología , Animales , Antineoplásicos/farmacología , División Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Ratones , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Células Tumorales Cultivadas , Xantenos/farmacologíaRESUMEN
5,6-dimethylxanthenone-4-acetic acid, a novel antivascular anticancer drug, has completed Phase I clinical trial. Its actions in mice include tumour necrosis factor induction, serotonin release, tumour blood flow inhibition, and the induction of tumour haemorrhagic necrosis and regression. We have used mice with a targeted disruption of the tumour necrosis factor receptor-1 gene as recipients for the colon 38 carcinoma to determine the role of tumour necrosis factor signalling in the action of 5,6-dimethylxanthenone-4-acetic acid. The pharmacokinetics of 5,6-dimethylxanthenone-4-acetic acid, as well as the degree of induced plasma and tissue tumour necrosis factor, were similar in tumour necrosis factor receptor-1(-/-) and wild-type mice. However, the maximum tolerated dose of 5,6-dimethylxanthenone-4-acetic acid was considerably higher in tumour necrosis factor receptor-1(-/-) mice (>100 mg kg(-1)) than in wild-type mice (27.5 mg kg(-1)). The antitumour activity of 5,6-dimethylxanthenone-4-acetic acid (25 mg kg(-1)) was strongly attenuated in tumour necrosis factor receptor-1(-/-) mice. However, the reduced toxicity in tumour necrosis factor receptor-1(-/-) mice allowed the demonstration that at a higher dose (50 mg kg(-1)), 5,6-dimethylxanthenone-4-acetic acid was curative and comparable in effect to that of a lower dose (25 mg kg(-1)) in wild-type mice. The 5,6-dimethylxanthenone-4-acetic acid -induced rise in plasma 5-hydroxyindoleacetic acid, used to reflect serotonin production in a vascular response, was larger in colon 38 tumour bearing than in non-tumour bearing tumour necrosis factor receptor-1(-/-) mice, but in each case the response was smaller than the corresponding response in wild-type mice. The results suggest an important role for tumour necrosis factor in mediating both the host toxicity and antitumour activity of 5,6-dimethylxanthenone-4-acetic acid, but also suggest that tumour necrosis factor can be replaced by other vasoactive factors in its antitumour action, an observation of relevance to current clinical studies.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias del Colon/tratamiento farmacológico , Factor de Necrosis Tumoral alfa/fisiología , Xantenos/uso terapéutico , Xantonas , Animales , Antígenos CD/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Relación Dosis-Respuesta a Droga , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Necrosis , Trasplante de Neoplasias , Receptores del Factor de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral , Serotonina/metabolismo , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismo , Xantenos/farmacocinéticaRESUMEN
5,6-Dimethylxanthenone-4-acetic acid, synthesised in this laboratory, reduces tumour blood flow, both in mice and in patients on Phase I trial. We used TUNEL (TdT-mediated dUTP nick end labelling) assays to investigate whether apoptosis induction was involved in its antivascular effect. 5,6-Dimethylxanthenone-4-acetic acid induced dose-dependent apoptosis in vitro in HECPP murine endothelial cells in the absence of up-regulation of mRNA for tumour necrosis factor. Selective apoptosis of endothelial cells was detected in vivo in sections of Colon 38 tumours in mice within 30 min of administration of 5,6-Dimethylxanthenone-4-acetic acid (25 mg x kg(-1)). TUNEL staining intensified with time and after 3 h, necrosis of adjacent tumour tissue was observed. Apoptosis of central vessels in splenic white pulp was also detected in tumour-bearing mice but not in mice without tumours. Apoptosis was not observed in liver tissue. No apoptosis was observed with the inactive analogue 8-methylxanthenone-4-acetic acid. Positive TUNEL staining of tumour vascular endothelium was evident in one patient in a Phase I clinical trial, from a breast tumour biopsy taken 3 and 24 h after infusion of 5,6-Dimethylxanthenone-4-acetic acid (3.1 mg x m(-2)). Tumour necrosis and the production of tumour tumour necrosis factor were not observed. No apoptotic staining was seen in tumour biopsies taken from two other patients (doses of 3.7 and 4.9 mg x m(-2)). We conclude that 5,6-Dimethylxanthenone-4-acetic acid can induce vascular endothelial cell apoptosis in some murine and human tumours. The action is rapid and appears to be independent of tumour necrosis factor induction.
Asunto(s)
Adenocarcinoma/irrigación sanguínea , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/irrigación sanguínea , Neoplasias del Colon/irrigación sanguínea , Endotelio Vascular/patología , Neoplasias Ováricas/irrigación sanguínea , Xantenos/uso terapéutico , Xantonas , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Apoptosis/fisiología , Northern Blotting , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Células Cultivadas , Quimiocina CXCL10 , Quimiocinas CXC/metabolismo , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Femenino , Humanos , Interferones/metabolismo , Ratones , Ratones Endogámicos C57BL , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
5,6-dimethylxanthenone-4-acetic acid (DMXAA), currently in phase I trials, demonstrates excellent activity against transplantable murine tumours with established vasculature. The induction of cytokines, particularly of tumour necrosis factor (TNF), appears to be critical to its action. We investigated TNF induction by DMXAA in cultured human peripheral blood leucocytes (HPBL). TNF was measured by an enzyme-linked immunosorbent assay after 8 h, and NF-kappaB induction by electrophoretic mobility shift assays (EMSA) after 2 h. DMXAA (800 microg/ml) had no effect alone on TNF production but augmented, by up to 4-fold, the ability of bacterial lipopolysaccharide (LPS) to induce TNF. Previously reported results showing TNF production by DMXAA alone were traced to the presence in an earlier batch of DMXAA of a small amount of LPS, the action of which could be blocked by polymyxin B. DMXAA stimulated TNF production by deacylated LPS, which alone had little effect. An antibody (MEM-18) to the CD14 receptor, while blocking the induction of TNF by LPS, enabled DMXAA to both synthesise TNF and induce NF-kappaB. The structurally related drug, flavone acetic acid (FAA), did not induce TNF or synergise with anti-CD14 antibody. DMXAA strongly augmented the ability of suboptimal concentrations of interleukin-1 (IL-1) (25 ng/ml), okadaic acid (OA) (20 ng/ml) and phorbol-12-myristate-13-acetate (PMA) (5 ng/ml) to induce TNF production, suggesting that it affects multiple pathways converging on NF-kappaB activation. Sodium salicylate, a drug reported to inhibit the beta-subunit of IkappaB kinase (IKK), appeared to competitively inhibit TNF production by DMXAA in the presence of anti-CD14 antibody. Taken together, the results indicate DMXAA acts in vitro on HPBL to co-stimulate TNF production by a wide variety of agents, and suggests that IKK is the target that mediates this action.
Asunto(s)
Antineoplásicos/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Factor de Necrosis Tumoral alfa/biosíntesis , Xantenos/farmacología , Xantonas , Técnicas de Cultivo de Célula , Citocinas/inmunología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática , Humanos , Leucocitos Mononucleares/inmunología , Receptores de Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , FN-kappa B/metabolismo , Ácido Ocadaico/farmacología , Ácido Salicílico/farmacología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
BACKGROUND: DMXAA (5,6-dimethylxanthenone-4-acetic acid) is a new drug synthesized in this laboratory and currently in phase I clinical trial. In mice it acts as an antivascular drug, selectively inhibiting tumour blood flow and inducing tumour haemorrhagic necrosis with resultant tumour regression. It also induces the synthesis of tumour necrosis factor (TNF), nitric oxide and serotonin. Cyproheptadine, a type 2 serotonin receptor antagonist, is known to reduce the degree of tumour necrosis-induced TNF in mice. We investigated the pharmacological interaction between a suboptimal dose of DMXAA (20 mg/kg) and cyproheptadine (20 mg/ kg) using mice with Colon 38 tumours that are sensitive to DMXAA. METHODS: Mice with or without tumours were treated with DMXAA and/or cyproheptadine. Concentrations of plasma and tissue DMXAA and the serotonin metabolite 5-hydroxyindoleacetic acid were measured by high performance liquid chromatography. TNF concentrations were measured by ELISA. RESULTS: While DMXAA alone (20 mg/kg) showed little or no antitumour activity, coadministration with cyproheptadine was curative in four of five mice. DMXAA half-lives in plasma and tumour tissue were increased 5.1- and 5.6-fold, respectively, and the appearance of DMXAA glucuronides in bile was almost completely inhibited for up to 4 h. Serum TNF was low and unchanged by cyproheptadine, and plasma concentrations of the serotonin metabolite 5-hydroxyindoleacetic acid were also not substantially changed. CONCLUSION: The augmentation by cyproheptadine of the induction of tumour response to DMXAA reflects a pharmacological interaction, leading to increased plasma and tumour half-lives, and to reduced excretion. However, serum TNF concentrations were not increased, suggesting that the increased anti-tumour effects are mediated by an increased local tumour response, arising from the extended tumour DMXAA concentrations.
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Antineoplásicos/farmacología , Neoplasias del Colon/tratamiento farmacológico , Ciproheptadina/farmacología , Antagonistas de la Serotonina/farmacología , Xantenos/farmacología , Xantonas , Animales , Antineoplásicos/farmacocinética , Neoplasias del Colon/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Quimioterapia Combinada , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Distribución Tisular , Xantenos/farmacocinéticaRESUMEN
PURPOSE: Coadministration of thalidomide, cyproheptadine or diclofenac has been shown to increase the area under the plasma concentration-time curve (AUC) of the novel antitumour agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) in mice. The aim of this study was to further investigate these pharmacokinetic DMXAA-drug interactions in the rat model. METHODS: The effects of coadministration of L-thalidomide, cyproheptadine or diclofenac on the pharmacokinetics of DMXAA were investigated in male Wistar Kyoto rats. The effects of L-thalidomide, cyproheptadine and diclofenac on microsomal metabolism and plasma protein binding of DMXAA were also investigated. RESULTS: No significant alteration in the plasma concentration profile for DMXAA was observed following L-thalidomide pretreatment in rats. In contrast, when combined with diclofenac or cyproheptadine, the plasma AUC of DMXAA was significantly (P<0.05) increased by 48% and 88% and the T1/2 by 36% and 107%, respectively, compared to controls. Both diclofenac and cyproheptadine at 500 microM caused a significant inhibition of DMXAA metabolism in rat liver microsomes. In contrast, L-thalidomide had no or little inhibitory effect on DMXAA metabolism in rat liver microsomes except for causing a 32% decrease in 6methylhydroxylation at 500 microM. None of the drugs had a significant effect on the plasma protein binding of DMXAA in the rat. CONCLUSION: These studies showed that coadministration of L-thalidomide did not alter the plasma DMXAA AUC in rats, in contrast to previous studies in mice, whereas diclofenac and cyproheptadine significantly reduced the plasma clearance of DMXAA in rats in a similar manner to their effect in mice. The cause of the species difference in the pharmacokinetic response to thalidomide by DMXAA is unknown, and indicates difficulties in predicting the outcome of such a combination in patients.
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Antineoplásicos/farmacocinética , Talidomida/farmacología , Xantenos/farmacocinética , Xantonas , Animales , Antineoplásicos/sangre , Ciproheptadina/farmacología , Diclofenaco/farmacología , Interacciones Farmacológicas , Glucurónidos/metabolismo , Hidroxilación , Masculino , Ratones , Microsomas Hepáticos/metabolismo , Ratas , Ratas Wistar , Talidomida/administración & dosificación , Xantenos/sangreRESUMEN
BACKGROUND: Previous studies have demonstrated that coadministration of L-thalidomide with the novel antitumour agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) results in an increased area under the plasma concentration-time curve (AUC) of DMXAA, suggesting an explanation for the observed increase in the antitumour activity. The aims of this study were to investigate the effects of L-thalidomide on the in vitro metabolism of DMXAA in mouse and human liver microsomes using diclofenac as positive control, to examine the effects of L-thalidomide and diclofenac on the plasma protein binding of DMXAA in vitro, and to investigate whether the in vivo interactions can be predicted from in vitro data, particularly in humans. METHODS: Mouse and human liver microsomes were used to investigate the effects of L-thalidomide and diclofenac on DMXAA metabolism. The resulting in vitro data were extrapolated to predict in vivo changes in DMXAA, which were then compared with the results of in vivo mouse pharmacokinetic interaction studies. The protein binding of DMXAA in mouse and human plasma was determined using ultrafiltration followed by HPLC. RESULTS: Diclofenac at 100 microM caused significant inhibition of glucuronidation (> 70%) and 6-methylhydroxylation (> 54%) of DMXAA in mouse and human liver microsomes. In vivo diclofenac (100 mg/kg i.p.) resulted in a 24% and 31% increase in the plasma DMXAA AUC, and a threefold increase in T1/2 (P < 0.05) in male and female mice, respectively. In contrast, L-thalidomide at 100 microM had no inhibitory effect on DMXAA metabolism in vitro in either species, except for a decrease of about 25% in 6-methylhydroxylation in mice. L-Thalidomide at 500 microM resulted in further significant decreases in 6-methylhydroxylation in mice (30-60%) and human (30%) microsomes. Coadministration of L-thalidomide in male mice resulted in a 23% increase in DMXAA AUC and a twofold increase in T1/2 (P < 0.05). Neither L-thalidomide nor diclofenac at 50 or 500 microM had any significant effect on the in vitro plasma protein binding of DMXAA (500 microM) in mouse or human plasma. Based on our in vitro inhibition studies, we predicted a 20% increase in DMXAA AUC in mice with concomitant diclofenac, but little or no effect (< 5%) with L-thalidomide. CONCLUSION: Both L-thalidomide and diclofenac increased the plasma DMXAA AUC in mice. In the case of diclofenac, this appeared to be due to direct competitive inhibition of DMXAA metabolism, but this mechanism does not appear to be appropriate for L-thalidomide. From the in vitro human inhibition studies, it appears unlikely that concurrent diclofenac will cause an increase in the plasma AUC of DMXAA in patients. However, the effect of L-thalidomide on DMXAA could not be readily predicted from the in vitro data. Our study demonstrated that a predictive model based on direct inhibition of metabolism is appropriate for diclofenac-DMXAA interactions, but is inappropriate for the prediction of L-thalidomide-DMXAA interactions in mice and humans in vivo.
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Antiinflamatorios no Esteroideos/farmacología , Antiinflamatorios no Esteroideos/farmacocinética , Antineoplásicos/farmacología , Antineoplásicos/farmacocinética , Diclofenaco/farmacología , Diclofenaco/farmacocinética , Teratógenos/farmacología , Teratógenos/farmacocinética , Talidomida/farmacología , Talidomida/farmacocinética , Xantenos/farmacología , Xantenos/farmacocinética , Xantonas , Animales , Proteínas Sanguíneas/metabolismo , Cromatografía Líquida de Alta Presión , Interacciones Farmacológicas , Femenino , Humanos , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Unión Proteica/efectos de los fármacosRESUMEN
The promise of cancer immunotherapy is that it will not only eradicate primary tumors but will generate systemic antitumor immunity capable of destroying distant metastases. A major problem that must first be surmounted relates to the immune resistance of large tumors. Here we reveal that immune resistance can be overcome by combining immunotherapy with a concerted attack on the tumor vasculature. The functionally related antitumor drugs 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and flavone acetic acid (FAA), which cause tumor vasculature collapse and tumor necrosis, were used to attack the tumor vasculature, whereas the T-cell costimulator B7.1 (CD80), which costimulates T-cell proliferation via the CD28 pathway, was used to stimulate antitumor immunity. The injection of cDNA (60-180 microg) encoding B7.1 into large EL-4 tumors (0.8 cm in diameter) established in C57BL/6 mice, followed 24 h later by i.p. administration of either DMXAA (25 mg/kg) or FAA (300 mg/kg), resulted in complete tumor eradication within 2-6 weeks. In contrast, monotherapies were ineffective. Both vascular attack and B7.1 immunotherapy led to up-regulation of heat shock protein 70 on stressed and dying tumor cells, potentially augmenting immunotherapy. Remarkably, large tumors took on the appearance of a wound that rapidly ameliorated, leaving perfectly healed skin. Combined therapy was mediated by CD8+ T cells and natural killer cells, accompanied by heightened and prolonged antitumor cytolytic activity (P < 0.001), and by a marked increase in tumor cell apoptosis. Cured animals completely rejected a challenge of 1 x 10(7) parental EL-4 tumor cells but not a challenge of 1 x 10(4) Lewis lung carcinoma cells, demonstrating that antitumor immunity was tumor specific. Adoptive transfer of 2 x 10(8) splenocytes from treated mice into recipients bearing established (0.8 cm in diameter) tumors resulted in rapid and complete tumor rejection within 3 weeks. Although DMXAA and B7.1 monotherapies are complicated by a narrow range of effective doses, combined therapy was less dosage dependent. Thus, a broad range of amounts of B7.1 cDNA were effective in combination with 25 mg/kg DMXAA. In contrast, DMXAA, which has a very narrow range of high active doses, was effective at a low dose (18 mg/kg) when administered with a large amount (180 microg) of B7.1 cDNA. Importantly, combinational therapy generated heightened antitumor immunity, such that gene transfer of B7.1 into one tumor, followed by systemic DMXAA treatment, led to the complete rejection of multiple untreated tumor nodules established in the opposing flank. These findings have important implications for the future direction and utility of cancer immunotherapies aimed at harnessing patients' immune responses to their own tumors.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Antígeno B7-1/inmunología , Inmunoterapia/métodos , Neovascularización Patológica/terapia , Xantonas , Animales , Antígeno B7-1/genética , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Terapia Combinada , Femenino , Flavonoides/administración & dosificación , Dosificación de Gen , Terapia Genética , Vectores Genéticos/genética , Células Asesinas Naturales/inmunología , Subgrupos Linfocitarios/inmunología , Linfoma/inmunología , Linfoma/patología , Linfoma/terapia , Ratones , Ratones Endogámicos C57BL , Linfocitos T Citotóxicos/inmunología , Neoplasias del Timo/irrigación sanguínea , Neoplasias del Timo/inmunología , Neoplasias del Timo/patología , Neoplasias del Timo/terapia , Xantenos/administración & dosificaciónRESUMEN
5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a drug synthesized in this laboratory that halts tumor blood flow and induces tumor hemorrhagic necrosis in transplantable murine tumors, is known to induce the synthesis of antiangiogenic cytokines in vitro. We have measured the induction of mRNA for modulators of angiogenesis in vivo and investigated whether DMXAA may also have an additional antiangiogenic action through the production of these cytokines. The genes for IFN-alpha and for interferon-inducible protein 10 (IP-10) were strongly induced in both spleen and Colon 38 tumor tissue after DMXAA treatment, whereas that for IFN-gamma was induced in spleen but not in tumor. Expression of mRNA for IFN-beta and for the p35 or the p40 subunits of interleukin 12 was not observed in either tissue. Splenic IP-10 mRNA induction was not a result of IFN-gamma production induced with DMXAA because spleen tissue from DMXAA-treated mice that lacked functional IFN-gamma receptors expressed similar amounts of IP-10 mRNA as those from wild-type mice. A single i.p. injection of DMXAA (20 mg/kg) was sufficient to reduce fibroblast growth factor-induced endothelial cell invasion of Matrigel implants in athymic nude mice by nearly 100%. The inactive analogue 8-methylxanthenone-4-acetic acid did not up-regulate the genes for IP-10 or IFNs and did not inhibit endothelial cell invasion. Antibodies to IP-10 reversed the inhibition of DMXAA of endothelial cell invasion by 58%; antibodies to tumor necrosis factor-alpha, IFN-gamma, and IFN-alpha reversed inhibition by 7%, 5%, and 0%, respectively. The data support the hypothesis that DMXAA, in addition to antivascular effects mediated by tumor necrosis factor-alpha, may have an antiangiogenic effect mediated largely by the induction of IP-10.
Asunto(s)
Antineoplásicos/farmacología , Quimiocinas CXC/biosíntesis , Neovascularización Fisiológica/efectos de los fármacos , Xantenos/farmacología , Xantonas , Animales , Anticuerpos/farmacología , Quimiocina CCL4 , Quimiocina CXCL10 , Quimiocinas CXC/genética , Quimiocinas CXC/inmunología , Colágeno , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Combinación de Medicamentos , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Femenino , Factor 2 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factor 2 de Crecimiento de Fibroblastos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Interferones/biosíntesis , Interferones/genética , Interleucina-12/biosíntesis , Interleucina-12/genética , Laminina , Proteínas Inflamatorias de Macrófagos/biosíntesis , Proteínas Inflamatorias de Macrófagos/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Proteoglicanos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Bazo/efectos de los fármacos , Bazo/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
A 16-year-old Chinese girl presented with AML-M5a. A bone marrow examination showed that the myeloblasts which were overwhelming the marrow contained giant granules (pseudo-Chediak-Higashi anomaly). Her karyotype showed a rare translocation t(10; 11)(p13; q14). Molecular delineation of the translocation breakpoints was not possible. Nonetheless, this case further demonstrates the morphological and phenotypic heterogeneity of acute leukaemia with this translocation. In this girl it was associated with disseminated intravascular coagulation.
Asunto(s)
Gránulos Citoplasmáticos/patología , Leucemia Monocítica Aguda/patología , Adolescente , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Células de la Médula Ósea/ultraestructura , Cromosomas Humanos Par 10/genética , Cromosomas Humanos Par 11/genética , Gránulos Citoplasmáticos/ultraestructura , Coagulación Intravascular Diseminada/patología , Femenino , Humanos , Cariotipificación , Leucemia Monocítica Aguda/genética , Microscopía Electrónica , Translocación GenéticaAsunto(s)
Cromosomas Humanos Par 13/genética , Cromosomas Humanos Par 1/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Preescolar , Cromosomas Humanos Par 17/genética , Humanos , Isocromosomas/genética , Masculino , Translocación GenéticaRESUMEN
BACKGROUND: 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), an investigative drug currently in clinical trial, acts on tumour vasculature through the induction of cytokines. Coadministration of thalidomide, a modulator of cytokine production, potentiates the antitumour activity of DMXAA against the murine Colon 38 carcinoma in mice. We wished to determine whether alteration of the pharmacokinetics of DMXAA by thalidomide could provide an explanation for this potentiation. RESULTS: Coadministration of thalidomide to Colon 38 tumour-bearing mice significantly (P < 0.05) increased the elimination half-life (t1/2) of DMXAA in plasma (413 micromol/l), liver (132 micromol/l), and spleen (77 micromol/l), and significantly (P < 0.05) increased DMXAA concentrations in Colon 38 tumour tissue (0.25-4.5 h). L-Thalidomide had a greater effect on DMXAA elimination (P < 0.01) than did D-thalidomide or the racemate. Coadministration of thalidomide increased the area under the concentration-time curve (AUC) of DMXAA by 1.8-fold in plasma, liver and spleen, and by 3.0-fold in tumour. Bile from mice given thalidomide and DMXAA contained substantially lower amounts of the glucuronide metabolite of DMXAA (DMXAA-G) than did bile from mice given DMXAA alone. CONCLUSION: Glucuronidation is a major excretory pathway for DMXAA in the mouse. Thalidomide, probably as the L-form, decreases the rate of elimination of DMXAA from plasma, spleen, liver and tumour by altering the rate of glucuronidation. The reduction in the elimination of DMXAA by thalidomide may lead to a selective increase in exposure of tumour tissue to drug, providing a basis for its potentiation of antitumour activity.
Asunto(s)
Antineoplásicos/farmacocinética , Neoplasias del Colon/metabolismo , Talidomida/farmacología , Xantenos/farmacocinética , Xantonas , Animales , Antineoplásicos/sangre , Área Bajo la Curva , Bilis/metabolismo , Neoplasias del Colon/sangre , Femenino , Semivida , Ratones , Ratones Endogámicos C57BL , Estereoisomerismo , Factores de Tiempo , Distribución Tisular/efectos de los fármacos , Xantenos/sangreRESUMEN
We describe the cytogenetic abnormalities in two cases of childhood hepatoblastoma. The first case was of fetal histology with squamous metaplasia, and cytogenetic study showed an add(5)(q31). Although an association between hepatoblastoma and familial adenomatous polyposis is recognized, the breakpoint in this case is distal to 5q21 and most probably does not involve the APC gene at that location. The second case was of macrotrabecular histology, and cytogenetic study showed an unbalanced translocation in the form of der(4)t(1;4)(q12;q34) in a hyperdiploid clone. Including our case, der(4)t(1;4)(q12;q34) has been recognized in four cases of hepatoblastoma, and it may be the first recurrent translocation in this tumor. Understanding the molecular mechanism and clinical significance of this translocation awaits analysis of more cases.
Asunto(s)
Aberraciones Cromosómicas , Hepatoblastoma/genética , Neoplasias Hepáticas/genética , Preescolar , Cromosomas Humanos Par 1 , Cromosomas Humanos Par 4 , Cromosomas Humanos Par 5 , Femenino , Hepatoblastoma/patología , Humanos , Lactante , Cariotipificación , Neoplasias Hepáticas/patología , MasculinoRESUMEN
We describe a case of T-lymphoblastic lymphoma (T-LBL) in a 13-year-old boy in which conventional cytogenetic analysis of lymph node tissue showed complex karyotypic aberrations including add(1p), add(2q), and multiple chromosomal deletions involving 5q, 7p, 7q, 13q, and 16q. Analysis by spectral karyotyping (SKY) refined add(1p) to a paracentric inversion of 1p, and add(2q) to an unbalanced translocation between chromosomes 2 and 4. The chromosomal deletions were simple deletions except del(5q), which was confirmed by SKY to be a cryptic unbalanced translocation between chromosomes 5 and 18. The present report illustrates that SKY technology is useful in identifying subtle translocations and resolving complex karyotypic aberrations in neoplastic disorders.