RESUMEN
Epigenetic therapy for solid tumors could benefit from an in vivo model that defines tumor characteristics of responsiveness and resistance to facilitate patient selection. Here we report that combining the histone deacetylase inhibitor entinostat with the demethylating agent vidaza profoundly affected growth of K-ras/p53 mutant lung adenocarcinomas engrafted orthotopically in immunocompromised nude rats by targeting and ablating pleomorphic cells that occupied up to 75% of the tumor masses. A similar reduction in tumor burden was seen with epigenetic therapy in K-ras or EGFR mutant tumors growing orthotopically. Increased expression of proapoptotic genes and the cyclin-dependent kinase inhibitor p21 was seen. Hundreds of genes were demethylated highlighted by the reexpression of polycomb-regulated genes coding for transcription factor binding proteins and the p16 gene, a key regulator of the cell cycle. Highly significant gene expression changes were seen in key regulatory pathways involved in cell cycle, DNA damage, apoptosis, and tissue remodeling. These findings show the promise for epigenetic therapy in cancer management and provide an orthotopic lung cancer model that can assess therapeutic efficacy and reprogramming of the epigenome in tumors harboring different genetic and epigenetic profiles to guide use of these drugs.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Azacitidina/administración & dosificación , Benzamidas/administración & dosificación , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Perfilación de la Expresión Génica , Inhibidores de Histona Desacetilasas/administración & dosificación , Humanos , Masculino , Piridinas/administración & dosificación , Ratas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Heparin has been shown to modify fundamental biologic processes ranging from blood coagulation and cell proliferation to fibrogenesis and asthma. The goal of this study was to identify specific or broad biologic responses of the rat lung to intratracheal instillation of heparin by targeted proteomic analysis. METHODS: Rats were given either aerosolized 500 microg heparin in 250 microl saline or saline alone. Lungs were harvested at 0, 24, or 96 hours post-treatment and isolated proteins analyzed by two-dimensional gel electrophoresis. Proteins which increased and decreased significantly in treated groups above controls were then selected for identification by mass spectrometry. RESULTS: Although heparin treatments resulted in a general reduction in cytosolic protein expression, there were significant increases within members of discrete groups of proteins. At 24 hours, proteins which function in cytoskeletal organization and in calcium signaling were up-regulated between 2- and 27-fold above baseline and untreated controls. Increased proteins include annexins V and VI, septin 2, capping G protein, actin-related protein 3, moesin, RhoGDP dissociation inhibitor, and calcyclin. A group of proteins relating to immune response and tumor suppressor function were either up-regulated (tumor suppressor p30/hyaluronic acid binding protein-1, Parkinson disease protein 7, proteosome 28 subunit/interferon-gamma inducible protein, and proteosome subunit macropain alpha-1) or strongly down-regulated (transgelin). At 96 hours, most proteins that had increased at 24 hours remained elevated but to a much lesser degree. CONCLUSION: These cumulative observations demonstrate that whole lung heparin treatment results in significant up-regulation of selected groups of proteins, primarily those related to cytoskeletal reorganization and immune function, which may prove to be relevant biomarkers useful in analysis of lung exposures/treatments as well as in system biology studies.
Asunto(s)
Anticoagulantes/administración & dosificación , Citoesqueleto/ultraestructura , Heparina/administración & dosificación , Pulmón/efectos de los fármacos , Pulmón/inmunología , Proteoma/efectos de los fármacos , Administración por Inhalación , Animales , Citoesqueleto/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Pulmón/ultraestructura , Proteómica/métodos , Ratas , Ratas Endogámicas F344 , Valores de ReferenciaRESUMEN
Several studies have found that smoking cigarettes is a risk factor for systemic lupus erythematosus (SLE). To examine this issue in a mouse model, we subjected pre-autoimmune MRL-lpr/lpr mice for 4 weeks to cigarette smoke to provide standardized smoke effluents equivalent to moderate or to heavy smoking habits for people. The spontaneous production of IgG anti-chromatin but not IgM anti-chromatin, anti-denatured DNA, or rheumatoid factor antibodies was lower in mice exposed to 250 mg/m3 particulates from mainstream smoke, and this suppression of autoimmunity was sustained for 8 weeks (p < 0.02). In contrast to control mice anti-chromatin activity in smoke-exposed mice began to increase in 16-week-old mice, reaching levels at 6 months that were two- to three-fold higher than controls for IgG (p < 0.03) and 10-fold higher for IgM (p < 0.001). There was no significant effect on total IgG or IgM. In newly diagnosed SLE patients, smoking was negatively correlated with IgG anti-DNA antibodies (p < 0.03). However, of nine patients who discontinued smoking prior to diagnosis, eight had elevated IgG anti-DNA compared to 29/79 never smokers and 9/31 smokers (p < 0.01 compared to former smokers). Inhaled cigarette smoke appears to have a long-lasting immunosuppressive effect on T-cell-dependent autoimmune responses, although autoantibodies increase to supra-elevated levels after the suppressive effect has abated.