RESUMEN
Triple-negative breast cancers (TNBC) are typically resistant to treatment, and strategies that build upon frontline therapy are needed. Targeting the murine double minute 2 (Mdm2) protein is an attractive approach, as Mdm2 levels are elevated in many therapy-refractive breast cancers. The Mdm2 protein-protein interaction inhibitor Nutlin-3a blocks the binding of Mdm2 to key signaling molecules such as p53 and p73α and can result in activation of cell death signaling pathways. In the present study, the therapeutic potential of carboplatin and Nutlin-3a to treat TNBC was investigated, as carboplatin is under evaluation in clinical trials for TNBC. In mutant p53 TMD231 TNBC cells, carboplatin and Nutlin-3a led to increased Mdm2 and was strongly synergistic in promoting cell death in vitro. Furthermore, sensitivity of TNBC cells to combination treatment was dependent on p73α. Following combination treatment, γH2AX increased and Mdm2 localized to a larger degree to chromatin compared with single-agent treatment, consistent with previous observations that Mdm2 binds to the Mre11/Rad50/Nbs1 complex associated with DNA and inhibits the DNA damage response. In vivo efficacy studies were conducted in the TMD231 orthotopic mammary fat pad model in NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (NSG) mice. Using an intermittent dosing schedule of combined carboplatin and Nutlin-3a, there was a significant reduction in primary tumor growth and lung metastases compared with vehicle and single-agent treatments. In addition, there was minimal toxicity to the bone marrow and normal tissues. These studies demonstrate that Mdm2 holds promise as a therapeutic target in combination with conventional therapy and may lead to new clinical therapies for TNBC.
Asunto(s)
Imidazoles/administración & dosificación , Neoplasias Pulmonares/tratamiento farmacológico , Piperazinas/administración & dosificación , Proteínas Proto-Oncogénicas c-mdm2/genética , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Animales , Carboplatino/administración & dosificación , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Ensayos Clínicos como Asunto , Daño del ADN/efectos de los fármacos , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Histonas/biosíntesis , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Células MCF-7 , Ratones , Metástasis de la Neoplasia , Proteínas Nucleares/genética , Neoplasias de la Mama Triple Negativas/patología , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
OBJECTIVES: The purpose of the present study was to develop and validate noninvasive bioluminescence imaging methods for differentially monitoring primary and abdominal metastatic tumor growth in mouse orthotopic models of pancreatic cancer. METHODS: A semiautomated maximum entropy segmentation method was implemented for the primary tumor region of interest, and a rule-based method for manually drawing a region of interest for the abdominal metastatic region was developed for monitoring tumor growth in orthotopic models of pancreatic cancer. The 2 region-of-interest methods were validated by having 2 observers independently segment Panc-1 tumors, and the results were compared with the number of mesenteric lymph node nodules and histopathologic assessment of liver metastases. The findings were extended to orthotopic tumors of the more metastatic MIA PaCa-2 and AsPC-1 cells where separate groups of animals were implanted with different numbers of cells. RESULTS: The results demonstrated that the segmentation methods were highly reliable, reproducible, and robust and allowed statistically significant discrimination in the growth rates of primary and abdominal metastatic tumors of different cell lines implanted with different numbers of cells. CONCLUSIONS: The present results demonstrate that primary tumors and abdominal metastatic foci in orthotopic pancreatic cancer models can be reliably quantified separately and noninvasively over time with bioluminescence imaging.
Asunto(s)
Neoplasias Abdominales/secundario , Neoplasias Hepáticas/secundario , Neoplasias Experimentales/patología , Imagen Óptica/métodos , Neoplasias Pancreáticas/patología , Animales , Automatización de Laboratorios , Línea Celular Tumoral , Proliferación Celular , Femenino , Xenoinjertos , Humanos , Procesamiento de Imagen Asistido por Computador , Mediciones Luminiscentes , Metástasis Linfática , Masculino , Ratones Endogámicos NOD , Micrometástasis de Neoplasia , Trasplante de Neoplasias , Reproducibilidad de los Resultados , Factores de Tiempo , Carga TumoralRESUMEN
Inhibition of vascular endothelial growth factor increases response rates to chemotherapy and progression-free survival in glioblastoma. However, resistance invariably occurs, prompting the urgent need for identification of synergizing agents. One possible strategy is to understand tumor adaptation to microenvironmental changes induced by antiangiogenic drugs and test agents that exploit this process. We used an in vivo glioblastoma-derived xenograft model of tumor escape in presence of continuous treatment with bevacizumab. U87-MG or U118-MG cells were subcutaneously implanted into either BALB/c SCID or athymic nude mice. Bevacizumab was given by intraperitoneal injection every 3 days (2.5 mg/kg/dose) and/or dichloroacetate (DCA) was administered by oral gavage twice daily (50 mg/kg/dose) when tumor volumes reached 0.3 cm(3) and continued until tumors reached approximately 1.5-2.0 cm(3). Microarray analysis of resistant U87 tumors revealed coordinated changes at the level of metabolic genes, in particular, a widening gap between glycolysis and mitochondrial respiration. There was a highly significant difference between U87-MG-implanted athymic nude mice 1 week after drug treatment. By 2 weeks of treatment, bevacizumab and DCA together dramatically blocked tumor growth compared to either drug alone. Similar results were seen in athymic nude mice implanted with U118-MG cells. We demonstrate for the first time that reversal of the bevacizumab-induced shift in metabolism using DCA is detrimental to neoplastic growth in vivo. As DCA is viewed as a promising agent targeting tumor metabolism, our data establish the timely proof of concept that combining it with antiangiogenic therapy represents a potent antineoplastic strategy.
Asunto(s)
Inhibidores de la Angiogénesis/administración & dosificación , Anticuerpos Monoclonales Humanizados/administración & dosificación , Ácido Dicloroacético/administración & dosificación , Resistencia a Antineoplásicos , Neoplasias/tratamiento farmacológico , Animales , Bevacizumab , Línea Celular Tumoral , Quimioterapia Combinada , Femenino , Humanos , Hipoxia , Ratones , Ratones SCID , Neoplasias/patología , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Humanized bone-marrow xenograft models that can monitor the long-term impact of gene-therapy strategies will help facilitate evaluation of clinical utility. The ability of the murine bone-marrow microenvironment in NOD/SCID versus NOD/SCID/γ chain(null) mice to support long-term engraftment of MGMT(P140K)-transduced human-hematopoietic cells following alkylator-mediated in vivo selection was investigated. Mice were transplanted with MGMT(P140K)-transduced CD34(+) cells and transduced cells selected in vivo. At 4 months after transplantation, levels of human-cell engraftment, and MGMT(P140K)-transduced cells in the bone marrow of NOD/SCID versus NSG mice varied slightly in vehicle- and drug-treated mice. In secondary transplants, although equal numbers of MGMT(P140K)-transduced human cells were transplanted, engraftment was significantly higher in NOD/SCID/γ chain(null) mice compared to NOD/SCID mice at 2 months after transplantation. These data indicate that reconstitution of NOD/SCID/γ chain(null) mice with human-hematopoietic cells represents a more promising model in which to test for genotoxicity and efficacy of strategies that focus on manipulation of long-term repopulating cells of human origin.
RESUMEN
Virtual screening targeting the urokinase receptor (uPAR) led to (±)-3-(benzo[d][1,3]dioxol-5-yl)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-4-phenylbutan-1-amine 1 (IPR-1) and N-(3,5-dimethylphenyl)-1-(4-isopropylphenyl)-5-(piperidin-4-yl)-1H-pyrazole-4-carboxamide 3 (IPR-69). Synthesis of an analogue of 1, namely, 2 (IPR-9), and 3 led to breast MDA-MB-231 invasion, migration and adhesion assays with IC(50) near 30 µM. Both compounds blocked angiogenesis with IC(50) of 3 µM. Compounds 2 and 3 inhibited cell growth with IC(50) of 6 and 18 µM and induced apoptosis. Biochemical assays revealed leadlike properties for 3, but not 2. Compound 3 administered orally reached peak concentration of nearly 40 µM with a half-life of about 2 h. In NOD-SCID mice inoculated with breast TMD-231 cells in their mammary fat pads, compound 3 showed a 20% reduction in tumor volumes and less extensive metastasis was observed for the treated mice. The suitable pharmacokinetic properties of 3 and the encouraging preliminary results in metastasis make it an ideal starting point for next generation compounds.