RESUMEN
The aim was to determine the potential of the allosteric mammalian target of rapamycin inhibitor, everolimus, to act in combination with cytotoxic anticancer compounds in vitro and in vivo. A concomitant combination in vitro showed no evidence of antagonism, but enhanced the antiproliferative effects (additive to synergistic) with cisplatin, doxorubicin, 5-fluorouracil, gemcitabine, paclitaxel, and patupilone. Everolimus (1-5 mg/kg/d orally) was evaluated for antitumor activity in vivo alone or in combination with suboptimal cytotoxic doses using athymic nude mice bearing subcutaneous human H-596 lung, KB-31 cervical, or HCT-116 colon tumor xenografts. Everolimus monotherapy was very well tolerated and caused inhibition of tumor growth, rather than regression, and this was associated with a dose-dependent decline in tumor pS6 levels, a key downstream protein of mammalian target of rapamycin. At the doses used, the cytotoxics inhibited tumor growth and caused tolerable body-weight loss. Concomitant combinations of cisplatin, doxorubicin, paclitaxel, or patupilone with everolimus produced cooperative antitumor effects, in some cases producing regressions without clinically significant increases in toxicity. In contrast, combinations with gemcitabine and 5-fluorouracil were less well tolerated. Alternative administration schedules were tested for cisplatin, gemcitabine, or paclitaxel combined with everolimus: these did not dramatically affect cisplatin or gemcitabine activity or tolerability but were antagonistic for paclitaxel. Everolimus showed promising maintenance activity after treatment with doxorubicin or paclitaxel ceased. Overall, the results confirm that everolimus is an effective, well-tolerated suppressor of experimental human tumor growth, and although it did not show strong potentiation of efficacy, antitumor activity in vivo was increased without marked increases in toxicity, supporting clinical use of everolimus as a partner for conventional cytotoxics.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/administración & dosificación , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Relación Dosis-Respuesta a Droga , Doxorrubicina/administración & dosificación , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Epotilonas/administración & dosificación , Everolimus , Femenino , Fluorouracilo/administración & dosificación , Células HCT116 , Humanos , Ratones , Ratones Desnudos , Paclitaxel/administración & dosificación , Paclitaxel/antagonistas & inhibidores , Sirolimus/administración & dosificación , Sirolimus/análogos & derivados , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
Early detection of tumors can significantly improve the outcome of tumor treatment. One of the most frequently asked questions in cancer imaging is how many cells can be detected non-invasively in a live animal. Although many factors limit such detection, increasing the light emission from cells is one of the most effective ways of overcoming these limitations. Here, we describe development and utilization of a lentiviral vector containing enhanced firefly luciferase (luc2) gene. The resulting single cell clones of the mouse mammary gland tumor (4T1-luc2) showed stable light emission in the range of 10,000 photons/sec/cell. In some cases individual 4T1-luc2 cells inserted under the skin of a nu/nu mouse could be detected non-invasively using a cooled CCD camera in some cases. In addition, we showed that only few cells are needed to develop tumors in these mice and tumor progression can be monitored right after the cells are implanted. Significantly higher luciferase activity in these cells allowed us to detect micrometastases in both, syngeneic Balb/c and nu/nu mice.
Asunto(s)
Diagnóstico por Imagen/métodos , Luciferasas/metabolismo , Mediciones Luminiscentes/métodos , Neoplasias Mamarias Experimentales/metabolismo , Animales , Línea Celular Tumoral , Femenino , Vectores Genéticos/genética , Lentivirus/genética , Luciferasas/genética , Mediciones Luminiscentes/instrumentación , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/diagnóstico , Neoplasias Mamarias Experimentales/genética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Sensibilidad y Especificidad , Factores de Tiempo , Transfección , Carga TumoralRESUMEN
PURPOSE: We have generated an EL1-luc/TAg transgenic mouse model that develops spontaneous and bioluminescent acinar cell carcinomas. We applied this model to noninvasively monitor tumor development and drug response. EXPERIMENTAL DESIGN: EL1-luc/TAg transgenic mice of 11 weeks of age were treated with rapamycin (5 mg/kg, i.p.) or vehicle for 6 to 12 weeks. Tumor development was monitored through bioluminescence imaging and necropsy at the study end point. RESULTS: EL1-luc/TAg transgenic mice showed pancreas-specific bioluminescence signal before tumor progression and produced increasing light emission from the onset of the pancreatic acinar cell carcinomas. The latency of tumor development ranged from 10 to >20 weeks of age in these mice. Progression of the primary acinar cell carcinoma was accompanied by emergence of metastatic lesions in the abdominal organs, including liver and gastrointestinal fat tissues. Rapamycin treatment suppressed tumor development. CONCLUSIONS: The EL1-luc/TAg mouse provides a noninvasive approach for monitoring spontaneous acinar cell carcinoma development and comprises a convenient tool for the evaluation of novel therapeutics against pancreatic cancers. Tumor growth suppression through inhibition of the mammalian target of rapamycin pathway further validates this model as clinically relevant.
Asunto(s)
Antibióticos Antineoplásicos/metabolismo , Carcinoma de Células Acinares/metabolismo , Monitoreo de Drogas , Páncreas/patología , Neoplasias Pancreáticas/patología , Animales , Antibióticos Antineoplásicos/uso terapéutico , Carcinoma de Células Acinares/tratamiento farmacológico , Modelos Animales de Enfermedad , Estudios Longitudinales , Mediciones Luminiscentes , Ratones , Ratones Transgénicos , Páncreas/efectos de los fármacos , Páncreas/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Sirolimus/metabolismo , Sirolimus/uso terapéuticoRESUMEN
07H239-A (1), a new eremophilane sesquiterpene from a marine-derived xylariaceous fungus, was isolated, characterized, and shown to be cytotoxic toward a variety of cancer cell lines, with some selectivity for a CCRFCEM leukemia line (IC(50) = 0.9 microg/mL).
Asunto(s)
Antineoplásicos/aislamiento & purificación , Ascomicetos/química , Naftalenos/aislamiento & purificación , Sesquiterpenos/aislamiento & purificación , Antineoplásicos/química , Antineoplásicos/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Ácidos Grasos Insaturados/química , Concentración 50 Inhibidora , Estructura Molecular , Naftalenos/química , Naftalenos/farmacología , Sesquiterpenos/química , Sesquiterpenos/farmacología , Células Tumorales CultivadasRESUMEN
Many proteins and pathways of pharmaceutical interest impinge on ubiquitin ligases or their substrates. The cyclin-dependent kinase (Cdk) inhibitor p27, for example, is polyubiquitylated in a cell cycle-dependent manner by a ubiquitin ligase complex containing the F-box protein Skp2. Regulated turnover of p27 is due, at least partly, to its phosphorylation by Cdk2 on threonine 187, which generates a Skp2-binding site. We made a p27-luciferase (p27Luc) fusion protein and show here that its abundance, like that of p27, is regulated by Skp2 in a cell cycle-dependent manner. As predicted, p27Luc levels increased after blocking Cdk2 activity with inhibitory proteins, peptides or small interfering RNA (siRNA). Accumulation of p27Luc in response to Cdk2 inhibitory drugs (flavopiridol and R-roscovitine) was demonstrable in human tumor cells in vivo using noninvasive bioluminescent imaging. In theory, the approach described here could be used to develop bioluminescent reporters for any drug target that directly or indirectly affects the turnover of a ubiquitin ligase substrate.
Asunto(s)
Quinasas CDC2-CDC28/metabolismo , Proteínas de Ciclo Celular/metabolismo , Diagnóstico por Imagen , Inhibidores Enzimáticos/metabolismo , Luciferasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/genética , Línea Celular , Trasplante de Células , Quinasa 2 Dependiente de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Genes Reporteros , Humanos , Luciferasas/genética , Mediciones Luminiscentes , Ratones , Ratones Desnudos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
We have synthesized a histone deacetylase inhibitor, NVP-LAQ824, a cinnamic hydroxamic acid, that inhibited in vitro enzymatic activities and transcriptionally activated the p21 promoter in reporter gene assays. NVP-LAQ824 selectively inhibited growth of cancer cell lines at submicromolar levels after 48-72 h of exposure, whereas higher concentrations and longer exposure times were required to retard the growth of normal dermal human fibroblasts. Flow cytometry studies revealed that both tumor and normal cells arrested in the G(2)-M phase of the cell cycle after compound treatment. However, an increased sub-G(1) population at 48 h (reminiscent of apoptotic cells) was observed only in the cancer cell line. Annexin V staining data supported our hypothesis that NVP-LAQ824 induced apoptosis in tumor and transformed cells but not in normal cells. Western blotting experiments showed an increased histone H3 and H4 acetylation level in NVP-LAQ824-treated cancer cells, suggesting that the likely in vivo target of NVP-LAQ824 was histone deacetylase(s). Finally, NVP-LAQ824 exhibited antitumor effects in a xenograft animal model. Together, our data indicated that the activity of NVP-LAQ824 was consistent with its intended mechanism of action. This novel histone deacetylase inhibitor is currently in clinical trials as an anticancer agent.
Asunto(s)
Antineoplásicos/toxicidad , Neoplasias del Colon/tratamiento farmacológico , Inhibidores de Histona Desacetilasas , Ácidos Hidroxámicos/toxicidad , Animales , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , División Celular/genética , Línea Celular Tumoral , Neoplasias del Colon/patología , Inhibidores Enzimáticos/toxicidad , Fluorouracilo/uso terapéutico , Histona Desacetilasas/aislamiento & purificación , Humanos , Cinética , Masculino , Ratones , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Trasplante HeterólogoRESUMEN
A series of N-hydroxy-3-phenyl-2-propenamides were prepared as novel inhibitors of human histone deacetylase (HDAC). These compounds were potent enzyme inhibitors, having IC(50)s < 400 nM in a partially purified enzyme assay. However, potency in cell growth inhibition assays ranged over 2 orders of magnitude in two human carcinoma cell lines. Selected compounds having cellular IC(50) < 750 nM were tested for maximum tolerated dose (MTD) and for efficacy in the HCT116 human colon tumor xenograft assay. Four compounds having an MTD > or = 100 mg/kg were selected for dose-response studies in the HCT116 xenograft model. One compound, 9 (NVP-LAQ824), had significant dose-related activity in the HCT116 colon and A549 lung tumor models, high MTD, and low gross toxicity. On the basis, in part, of these properties, 9 has entered human clinical trials in 2002.
Asunto(s)
Acetiltransferasas/antagonistas & inhibidores , Acrilamidas/síntesis química , Acrilamidas/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Ácidos Hidroxámicos/síntesis química , Ácidos Hidroxámicos/farmacología , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Animales , Peso Corporal/efectos de los fármacos , División Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Histona Acetiltransferasas , Humanos , Indicadores y Reactivos , Ratones , Ratones Desnudos , Conformación Molecular , Trasplante de NeoplasiasRESUMEN
Vitilevuamide, a bicyclic 13 amino acid peptide, was isolated from two marine ascidians, Didemnum cuculiferum and Polysyncranton lithostrotum. Vitilevuamide was cytotoxic in several human tumor cell lines, with LC(50) values ranging from 6 to 311nM, and analysis in a 25-cell line panel revealed a weak correlation with several taxol analogs. Vitilevuamide was strongly positive in a cell-based screen for inhibitors of tubulin polymerization. Vitilevuamide at 9 microg/mL (5.6 microM) had an effect equivalent to the maximal effect of colchicine at 25 microg/mL (62.5 microM). Vitilevuamide was active in vivo against P388 lymphocytic leukemia, increasing the lifespan of leukemic mice 70% at 30 microg/kg. We hypothesized that at least part of the cytotoxic mechanism of vitilevuamide was due to its inhibition of tubulin polymerization. Vitilevuamide was found to inhibit polymerization of purified tubulin in vitro, with an IC(50) value of approximately 2 microM. Cell cycle analysis showed that vitilevuamide arrested cells in the G(2)/M phase with 78% of treated cells tetraploid after 16hr. Therefore, vitilevuamide was tested for its ability to inhibit binding of known tubulin ligands. Vitilevuamide exhibited non-competitive inhibition of vinblastine binding to tubulin. Colchicine binding to tubulin was stabilized in the presence of vitilevuamide in a fashion similar to vinblastine. Dolastatin 10 binding was unaffected by vitilevuamide at low concentrations, but inhibited at higher ones. GTP binding was also found to be weakly affected by the presence of vitilevuamide. These results suggest the possibility that vitilevuamide inhibits tubulin polymerization via an interaction at a unique site.