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.
Asunto(s)
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.
Asunto(s)
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.
Asunto(s)
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
An established in vitro cell line (LLTC), originally derived from the Lewis lung carcinoma (LL), was found to have lost sensitivity to the C-nucleoside antitumor agent tiazofurin (NSC-286193; 2-beta-D-ribofuranosylthiazole-4-carboxamide) both in vitro and in vivo. A new in vitro cell line (LLAK) was derived from LL and compared to LLTC in its growth properties and sensitivity to tiazofurin. LLAK resembled the in vivo tumor in having both a high S-phase fraction and a high rate of cell death at high cell density. In continuous drug exposure growth inhibition assays, the concentration of tiazofurin required to reduce the number of cells in a culture by 50% with respect to control cultures was 0.51 microM for LLAK, 2.6 microM for LLTC, and greater than 10 microM for a range of human cancer cell lines. In cytotoxicity assays involving a 2-hour drug exposure followed by clonogenic assay, tiazofurin was more toxic to LLAK cells than to LLTC cells or L1210 murine leukemia cells, consistent with its high in vivo activity against LL. MM-96 human melanoma cells were highly resistant. Flow cytometry studies indicated that tiazofurin selectively depleted the LLAK cell population of S- and G2-phase cells. In one experiment involving 16 consecutive in vitro passages of LLAK in the absence of tiazofurin, a new line emerged that was resistant to tiazofurin in the clonogenic assay. The results demonstrate the spontaneous emergence of resistance from a tiazofurin-sensitive cell line. If similar processes occur during adaptation of human tumor cells to culture, this may explain the finding of low activity of tiazofurin toward a range of human tumor cell lines.
Asunto(s)
Ensayo de Unidades Formadoras de Colonias , Neoplasias Pulmonares/tratamiento farmacológico , Ribavirina/uso terapéutico , Ribonucleósidos/uso terapéutico , Ensayo de Tumor de Célula Madre , Animales , Línea Celular , Células Clonales/efectos de los fármacos , Resistencia a Medicamentos , Citometría de Flujo , Humanos , Ratones , Ratones Endogámicos C57BL , Ribavirina/análogos & derivadosRESUMEN
Activated peritoneal macrophages, obtained from mice pretreated with Bacillus Calmette-Guérin, after exposure in vitro to flavone-8-acetic acid (FAA; NSC 347512) at a concentration of 890 microM, produce nitrite (3.7 nmol/10(6) cells), as measured 20 h later by the Griess reaction. Stimulation of nitrite production was inhibited at least 90% by NG-monomethylarginine (125 microM), suggesting that nitrite was formed via nitric oxide as a product of arginine metabolism. Stimulation was only partially inhibited by dexamethasone (0.1 microM). The ability of xanthenone-4-acetic acid (XAA) and three of its analogues to stimulate nitrite production was also investigated. 5,6-Dimethyl-XAA stimulated nitrite production (12.6 nmol/10(6) cells) at an optimal concentration of 80 microM, 8-methyl-XAA was without effect, and XAA and 5-methyl-XAA showed intermediate activity. The optimal in vitro drug concentrations for stimulation by FAA, XAA, and active XAA analogues correlated with the optimal in vivo dose required for the induction of either hemorrhagic necrosis or growth delay of s.c. Colon 38 tumors. These results strongly imply that FAA and active XAA derivatives function as low molecular weight stimulators of nitric oxide formation in macrophages, possibly acting on the same differentiation pathway as do endotoxin and tumor necrosis factor alpha. We suggest that nitric oxide, which is known to be toxic to tumor cells, contributes to the cytotoxic action of FAA and its analogues.
Asunto(s)
Antineoplásicos/farmacología , Flavonoides/farmacología , Macrófagos/metabolismo , Óxido Nítrico/metabolismo , Xantenos/farmacología , Animales , Arginina/análogos & derivados , Arginina/farmacología , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Dexametasona/farmacología , Ratones , Trasplante de Neoplasias , Nitratos , Ácido Nítrico , omega-N-MetilargininaRESUMEN
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
The investigational antitumor agent, 5,6-dimethylxanthenone-4-acetic acid (5,6-MeXAA; NSC 640488) induced greater expression of tumor necrosis factor-alpha (TNF-alpha) mRNA in murine spleen cells in vivo at its optimal dose of 27.5 mg/kg than flavone acetic acid (FAA; NSC 347512) at its optimal dose of 220 mg/kg. Up-regulation of TNF-alpha mRNA was obtained using 5,6-MeXAA in vitro in cultures of murine splenocytes, the murine J774 macrophage cell line, and the human HL-60 myelomonocytic leukemia cell line. Maximal induction occurred at a 5,6-MeXAA concentration of 200 micrograms/ml for both murine J774 and human HL-60 cells. A direct comparison of FAA and 5,6-MeXAA (100-600 micrograms/ml) to stimulate TNF-alpha mRNA in HL-60 cells showed activity by 5,6-MeXAA at all doses but minimal activity with FAA. The results demonstrate that 5,6-MeXAA is equally potent in up-regulating TNF-alpha mRNA in human and murine cells of the monocyte/macrophage lineage, whereas FAA has demonstrable activity in murine cells only. The results suggest that 5,6-MeXAA would be a more active clinical agent than FAA because TNF-alpha induction appears to be a critical factor in the antitumor effects of this class of compounds.
Asunto(s)
Antineoplásicos/farmacología , ARN Mensajero/biosíntesis , Factor de Necrosis Tumoral alfa/genética , Xantenos/farmacología , Xantonas , Animales , Humanos , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Especificidad de la EspecieRESUMEN
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
The antitumor agent flavone-8-acetic acid (FAA) is remarkable because it induces hemorrhagic necrosis, altered tumor blood flow, and cytokine synthesis. We show here that FAA and structurally related analogues increase plasma nitrite plus nitrate (NO2-/NO3-) levels in mice. Dose-dependent increases in plasma NO2-/NO3- concentrations, which reached maximum levels at 12 h, were found following administration of FAA. Furthermore, the presence of a palpable s.c. Colon 38 tumor significantly enhanced the response. Tumor-dependent increases were also observed with the active FAA analogues xanthenone-4-acetic acid, 5-methyl XAA, and 5,6-dimethyl XAA, while the inactive analogue 8-methyl XAA failed to increase plasma NO2-/NO3- concentrations substantially above basal levels. Increased plasma NO2-/NO3- levels were also observed in response to endotoxin (100 micrograms/mouse) and to recombinant human tumor necrosis factor alpha (4 to 16 micrograms/mouse). NO2-/NO3- levels may signify nitric oxide production as a result of stimulation of the L-arginine-dependent pathway in activated macrophages. The tumor dependence of the response may reflect the immunological stimulus imposed by tumor implantation. A clear relationship was found between increased plasma NO2-/NO3- levels and tumor growth delays induced by FAA and xanthenone-4-acetic acid analogues. It is suggested that nitric oxide may contribute to tumor cell death by two mechanisms, alteration of blood flow contributing to tumor ischemia and direct tumor cell killing. Plasma NO2-/NO3- concentrations may be a sensitive indication of the antitumor response to this class of compounds.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Neoplasias del Colon/tratamiento farmacológico , Flavonoides/uso terapéutico , Nitratos/sangre , Xantonas , Animales , Relación Dosis-Respuesta a Droga , Metilación , Ratones , Ratones Endogámicos , Nitritos/sangre , Xantenos/uso terapéuticoRESUMEN
5,6-Dimethylxanthenon-4-acetic acid (DMXAA) is a new antitumor drug currently undergoing clinical trial. Administration of DMXAA to mice with tumors leads to cessation of tumor blood flow and the onset of tumor hemorrhagic necrosis, accompanied by the production of the cytokine tumor necrosis factor (TNF). Previous studies have shown that DMXAA induces both tumor and host cells to synthesize TNF and that induced intratumoral TNF production correlates with the antitumor activity of DMXAA. To explore the hypothesis that TNF production by tumor cells contributed to the induction of hemorrhagic necrosis by DMXAA, TNF-/- (C57Bl/6 background) mice were used as recipients for the s.c. implantation of (TNF positive) colon 38 adenocarcinoma. Tumors removed 24 h after treatment with DMXAA (66 or 100 micromol/kg) were found to be hemorrhagic and necrotic. Cells expressing TNF mRNA in tumors removed 2 h after treatment with DMXAA (160 micromol/kg) were found by in situ hybridization to be comparable in frequency and distribution with those in tumors from C57Bl/6 TNF-positive mice. However, the amount of TNF protein extracted from tumors from TNF knockout mice was lower than that from TNF-positive mice. Spleen and liver tissue from TNF knockout mice, in contrast to that from TNF-positive mice, produced no TNF mRNA. TNF protein was undetectable in liver and spleen tissue from TNF knockout mice, but was evident in tissue from TNF-positive mice. These results confirm that DMXAA has the novel ability of inducing tumors to synthesize TNF in situ.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Antineoplásicos/uso terapéutico , Neoplasias del Colon/tratamiento farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas de Neoplasias/biosíntesis , Neovascularización Patológica/tratamiento farmacológico , Factor de Necrosis Tumoral alfa/biosíntesis , Xantenos/uso terapéutico , Xantonas , Adenocarcinoma/irrigación sanguínea , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Antineoplásicos/farmacología , Neoplasias del Colon/irrigación sanguínea , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Hemorragia/inducido químicamente , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Necrosis , Proteínas de Neoplasias/genética , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , Factor de Necrosis Tumoral alfa/deficiencia , Factor de Necrosis Tumoral alfa/genética , Xantenos/farmacologíaRESUMEN
The selective induction of tumor vascular collapse represents an exciting approach to cancer treatment. However, clinical evaluation of tumor necrosis factor-alpha (TNF), an agent that accomplishes this goal, has been limited by systemic toxicity, and clinical approaches using bacterial components to induce TNF production have also been disappointing. Our laboratory has developed synthetic low molecular weight inducers of TNF, including 5,6-dimethylxanthenone-4-acetic acid (DMXAA), as an alternative strategy. DMXAA induces rapid vascular collapse in transplantable murine tumors and induces TNF synthesis in vitro in both murine and human systems. We show here that the extent of DMXAA-induced TNF synthesis is greater in tumors than that in the spleen, liver, or serum. As shown by in situ hybridization studies of the murine Colon 38 tumor, DMXAA induced tumor as well as host cells to express TNF mRNA. The distribution of cells containing TNF mRNA in tumor tissues after DMXAA administration contrasted significantly with that obtained after lipopolysaccharide (LPS) treatment, although splenic and hepatic tissues showed a similar distribution of TNF mRNA-positive cells. In the Colon 38 tumor, the action of LPS was limited to host cells in the periphery of the vessels. DMXAA treatment induced 7-fold higher peak TNF levels in tumor than in serum. In contrast, LPS treatment induced 9-fold higher TNF levels in serum than in tumor. DMXAA induced 35-fold higher TNF activity in the Colon 38 tissue than did LPS. One ovarian, one squamous, and three melanoma human tumor xenografts implanted in athymic nude mice expressed TNF mRNA of human and murine origin in response to DMXAA, confirming that DMXAA can activate both host and tumor cells. The use of low molecular weight agents to induce TNF synthesis in situ in the tumor represents a novel approach to TNF-mediated therapy of cancers.
Asunto(s)
Antineoplásicos/farmacología , Factor de Necrosis Tumoral alfa/biosíntesis , Xantenos/farmacología , Xantonas , Animales , Carcinoma de Células Escamosas/metabolismo , Femenino , Humanos , Lipopolisacáridos/farmacología , Hígado/metabolismo , Melanoma/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias Ováricas/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/metabolismo , Bazo/metabolismo , Estimulación Química , Trasplante HeterólogoRESUMEN
A case of Ph-negative M-BCR rearranged eosinophilic leukaemia with clonal cytogenetic abnormalities is presented. In addition to involvement of the short arm of chromosome 12 (12p12?13), the malignant nature of the eosinophils was confirmed by the demonstration of trisomy 8 by in situ hybridization.
Asunto(s)
Cromosomas Humanos Par 8 , Proteínas de Fusión bcr-abl/genética , Reordenamiento Génico/genética , Síndrome Hipereosinofílico/genética , Cromosoma Filadelfia , Trisomía/genética , Adulto , Cromosomas Humanos Par 12 , Cromosomas Humanos Par 17 , ADN de Neoplasias/genética , Eosinófilos/fisiología , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Masculino , Monosomía/genéticaRESUMEN
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
Xanthenone-4-acetic acid (XAA) resembles flavone acetic acid (FAA) in its effects on solid tumours in mice. The activity of methyl-substituted XAA derivatives in vitro was determined using 18 h 51Cr-release assays, continuous exposure growth inhibition assays and stimulation of tumouricidal activity of cultured murine resident peritoneal macrophages. The macrophage assay identified the high biological activity and dose potency of 5-MeXAA in vivo, and was the most accurate in vitro predictor of the ability of congeners to induce either haemorrhagic necrosis of subcutaneous Lewis lung and colon 38 tumours or splenic natural killer activity. In vitro immune stimulation may be more appropriate than direct cytotoxicity for screening compounds with indirect mechanisms of antitumour activity.
Asunto(s)
Antineoplásicos/farmacología , Xantenos/farmacología , Xantonas , Animales , Supervivencia Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Activación de Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Endogámicos , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Human and mouse bone marrow cells were cultured for 1 h in the presence of either the antileukaemia drug amsacrine or its 4-methyl,5-[N-methyl]carboxamide disubstituted analogue CI-921, before being plated in methylcellulose medium to determine the survival of granulocyte-macrophage colony forming units (CFU-GM). The drug concentration required for 50% reduction in survival was approx. 0.4 microM for both drugs and was similar for both human and mouse cells. A comparison of the two drugs was then made, at an added drug concentration of 0.5 microM, using cultured mouse L1210 and P388 leukaemia, Lewis lung carcinoma cell lines LLAK and LLTC, human Jurkat leukaemia, human histiocytic lymphoma U937 and human colon carcinoma SW620. The sensitivity of the mouse lines for amsacrine was in the order L1210 greater than P388 greater than LLAK greater than LLTC, similar to the in vivo sensitivity. The selectivity of CI-921 for L1210 versus bone marrow, and for LLAK versus L1210 or P388, was greater than that of amsacrine, again in keeping with its in vivo properties. The sensitivity of the human Jurkat and U937 lines for amsacrine was intermediate between that of L1210 and P388, while SW620 was resistant. The selectivity of CI-921 for Jurkat and U937 versus bone marrow was greater than that of amsacrine, suggesting that CI-921 could have additional advantages over amsacrine in the treatment of some tumours.
Asunto(s)
Amsacrina/análogos & derivados , Amsacrina/uso terapéutico , Médula Ósea/patología , Leucemia Experimental/tratamiento farmacológico , Animales , Antineoplásicos/uso terapéutico , Médula Ósea/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Leucemia L1210/tratamiento farmacológico , Ratones , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a novel antitumour agent currently undergoing clinical evaluation, appears to mediate its antitumour effects through immune modulation and the production of cytokines. We used mice with a targeted disruption of the interferon-gamma (IFN-gamma) receptor gene as a model to evaluate the role of the host response to IFN-gamma in the antitumour action of DMXAA on colon 38 tumours. A feature of the results was that while DMXAA treatment induced both IFN-gamma and tumour necrosis factor (TNF) in serum, the increase was > 20-fold higher in IFN-gamma R0/0 mice than in wild-type mice. In contrast, mRNA levels for IFN-gamma and TNF were similar in the two mouse strains, suggesting that the concentrations of these cytokines were controlled by a post-transcriptional mechanism. Serum nitrate levels, used as a measure of nitric oxide production, were increased by DMXAA, but to a similar extent in both strains of mice. Complete regressions of colon 38 tumours were obtained in response to DMXAA in the knockout mice, although the dose required for 100% cure was higher and the reduction in tumour volume occurred more slowly than in the wild-type counterparts. The results demonstrate that the host response to IFN-gamma is not essential for an anti-tumour response. Similar results were obtained in mice that were immunosuppressed by treatment with cyclosporin A before treatment with DMXAA. The results are consistent with the concept that the antitumour activity of DMXAA involves complex immunomodulation, probably with significant redundancy in contributing cytokines.
Asunto(s)
Neoplasias del Colon/inmunología , Interferón gamma/inmunología , Receptores de Interferón/inmunología , Xantenos/inmunología , Xantonas , Animales , Neoplasias del Colon/tratamiento farmacológico , Ciclosporina/farmacología , Ensayo de Inmunoadsorción Enzimática , Inmunosupresores/farmacología , Lipopolisacáridos/farmacología , Ratones , Óxido Nítrico/metabolismo , ARN Mensajero/metabolismo , ARN Neoplásico/metabolismo , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa , Xantenos/uso terapéutico , Receptor de Interferón gammaRESUMEN
Flavone-8-acetic acid (FAA) induces haemorrhagic necrosis and tumour regression in experimental tumours and induces natural killer (NK) activity. Xanthenone-4-acetic acid (XAA) forms the basis of a series of analogues of FAA which vary in antitumour potency. FAA, XAA and 15 XAA derivatives were tested for their ability to induce either NK activity in mouse spleens or haemorrhagic necrosis in mouse colon 38 tumours. Some derivatives were active in both assays (one at a dose 8-fold lower than that of FAA). When both assays were quantitated, a significant correlation (r = 0.85; P less than 0.001) was found. NK assays could be useful in screening compounds such as FAA and XAA analogues which appear to mediate their antitumour activity by biological response modification. Since tumour necrosis may not be mediated directly by NK cells, FAA and active XAA derivatives may exert pleiotropic effects that include NK induction and tumour necrosis by acting on host cells to release cytokines.
Asunto(s)
Neoplasias del Colon/terapia , Citotoxicidad Inmunológica/efectos de los fármacos , Células Asesinas Naturales/inmunología , Xantenos/farmacología , Animales , Neoplasias del Colon/inmunología , Neoplasias del Colon/patología , Relación Dosis-Respuesta Inmunológica , Factores Inmunológicos/uso terapéutico , Ratones , Ratones Endogámicos , Xantenos/uso terapéuticoRESUMEN
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
The new antitumour drug 5,6-dimethylxanthenone-4-acetic acid (5,6-MeXAA; NSC 640488) was 14-fold more potent than the investigational chemotherapeutic drug flavone-8-acetic acid (NSC 347512) in stimulating tumouricidal activity in cultures of resident murine peritoneal macrophages. The tumouricidal activity of thioglycollate-elicited and Bacillus Calmette-Guérin-primed macrophages was also significantly enhanced by 5,6-MeXAA. Stimulation of macrophage tumouricidal activity by 5,6-MeXAA was not affected by inhibitors of superoxide and nitric oxide production, but was reduced by cyclosporin A, an inhibitor of protein secretion. Inhibitors of neutral proteases had no effect. Cortisone, dexamethasone, indomethacin, dibutyryl cAMP, prostaglandin E2 and prostacyclin, but not prostaglandin F2 alpha, inhibited stimulation, suggesting the involvement of tumour necrosis factor-alpha (TNF). However, antibodies to TNF did not inhibit stimulation. The results suggest that 5,6-MeXAA acts on macrophages in a manner similar to that of endotoxin, utilizing a pathway which includes arachidonic acid metabolism and requiring cell-cell contact with target cells for a tumouricidal effect.