RESUMEN
As a growing number of patients with multiple myeloma (MM) respond to upfront therapies while eventually relapsing in a time frame that is often unpredictable, attention has increasingly focused on developing novel diagnostic criteria to also account for disease dissemination. Positron emission tomography/computed tomography (PET/CT) is often used as a noninvasive monitoring strategy to assess cancer cell dissemination, but because the uptake of the currently used radiotracer 18fluorodeoxyglucose (18F-FDG) is a function of the metabolic activity of both malignant and nonmalignant cells, the results frequently lack sufficient specificity. Radiolabeled antibodies targeting MM tissue may detect disease irrespective of cell metabolism. Hence, we conjugated the clinically significant CD38-directed human antibody daratumumab (Darzalex [Dara]) to the DOTA chelator and labeled it with the positron-emitting radionuclide copper 64 (64Cu; 64Cu-DOTA-Dara). Here, we show that 64Cu-DOTA-Dara can efficiently bind CD38 on the surface of MM cells and was mainly detected in the bones associated with tumor in a MM murine model. We also show that PET/CT based on 64Cu-DOTA-Dara displays a higher resolution and specificity to detect MM cell dissemination than does 18F-FDG PET/CT and was even more sensitive than were bioluminescence signals. We therefore have supporting evidence for using 64Cu-DOTA-Dara as a novel imaging agent for MM.
Asunto(s)
Anticuerpos Monoclonales , Radioisótopos de Cobre , Mieloma Múltiple/diagnóstico , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Animales , Anticuerpos Monoclonales/farmacocinética , Línea Celular Tumoral , Rastreo Celular/métodos , Radioisótopos de Cobre/farmacocinética , Semivida , Xenoinjertos , Humanos , Ratones , Mieloma Múltiple/metabolismo , Trasplante de Neoplasias , Trazadores RadiactivosRESUMEN
microRNA-146a (miR-146a) has been previously implicated as an essential molecular brake, preventing immune overreaction and malignant transformation by attenuating NF-κB signaling, putatively via repression of the Traf6 and Irak1 genes. The exact contribution of miR-146a-mediated silencing of these genes to the control of immune activation is currently unknown. Therefore, we defined the role of the miR-146a-Traf6 signaling axis in the regulation of immune homeostasis using a genetic epistasis analysis in miR-146a-/- mice. We have uncovered a surprising separation of functions at the level of miR-146a targets. Lowering the Traf6 gene dose and consequent attenuation of NF-κB activation rescued several significant miR-146a-/- phenotypes, such as splenomegaly, aberrant myeloproliferation, and excessive inflammatory responses. In contrast, decreasing Traf6 expression had no effect on the development of the progressive bone marrow failure phenotype, as well as lymphomagenesis in miR-146a-/- mice, indicating that miR-146a controls these biological processes through different molecular mechanisms.
Asunto(s)
Autoinmunidad , Células Madre Hematopoyéticas/citología , Inflamación/inmunología , MicroARNs/inmunología , Mielopoyesis , Neoplasias/inmunología , Factor 6 Asociado a Receptor de TNF/inmunología , Animales , Femenino , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/inmunología , Homeostasis , Humanos , Inflamación/genética , Inflamación/fisiopatología , Masculino , Ratones , MicroARNs/genética , Células Mieloides/citología , Células Mieloides/inmunología , Neoplasias/genética , Neoplasias/fisiopatología , Factor 6 Asociado a Receptor de TNF/genéticaRESUMEN
Substantial evidence suggests that 7,12-dimethylbenzanthracene (DMBA)-induced mammary carcinogenesis in mice mimics human breast cancer (BC) in many respects. Therefore, it has been used extensively to evaluate preventive and therapeutic agents for human BC. Mammary carcinogenesis induced by DMBA administration in female SENsitive to CARcinogen (SENCAR) mice was characterized by histopathological analysis of the mammary glands and alterations to the phosphatidylinositol 3-kinase/protein kinase B/cyclin-dependent kinase 1 (PI3K/Akt/CDK1) pathway. We recently reported that 2'-hydroxyflavanone (2HF) is a promising diet-derived chemotherapeutic agent that suppresses BC growth in vitro and in vivo by targeting a 76 kDa ral-interacting protein (RLIP). The objective of the current study was to investigate the synergistic anticarcinogenic effects of RLIP inhibition/depletion and 2HF in an in vivo model of DMBA-induced mammary carcinogenesis in SENCAR mice. Mice were given 2HF (50 mg/kg, bw, orally on alternate days), RLIP antibody (Rab; 5 mg/kg, bw, ip weekly), RLIP antisense (RAS; 5 mg/kg, b.w., ip weekly), or a combination of 2HF + Rab + RAS. Animals were monitored daily, and 7 days after the first appearance of moribund behavior, tissues were harvested for morphological and immunohistological analysis. Western blot analyses were performed to determine the expression of anti- and proapoptotic proteins in the mammary glands. Our results reveal that 2HF, RAS, and Rab significantly prevented the carcinogenic effects of DMBA administration in the mammary glands and other organs. Further, mice treated with a combination of 2HF + RAS + Rab exhibited no carcinogenic effect of DMBA as compared to either or the single agent-treated mice. This study demonstrates for the first time the anticarcinogenic effects of 2HF and RLIP inhibition/depletion in vivo in a novel DMBA-induced model of BC in SENCAR mice and provides the rationale for further clinical investigation.
Asunto(s)
9,10-Dimetil-1,2-benzantraceno/toxicidad , Anticarcinógenos/farmacología , Transformación Celular Neoplásica/patología , Flavanonas/farmacología , Proteínas Activadoras de GTPasa/antagonistas & inhibidores , Neoplasias Mamarias Experimentales/prevención & control , Animales , Proteína Quinasa CDC2/metabolismo , Modelos Animales de Enfermedad , Femenino , Proteínas Activadoras de GTPasa/genética , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos SENCAR , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Glucose homeostasis is a vital and complex process, and its disruption can cause hyperglycaemia and type II diabetes mellitus. Glucokinase (GK), a key enzyme that regulates glucose homeostasis, converts glucose to glucose-6-phosphate in pancreatic ß-cells, liver hepatocytes, specific hypothalamic neurons, and gut enterocytes. In hepatocytes, GK regulates glucose uptake and glycogen synthesis, suppresses glucose production, and is subject to the endogenous inhibitor GK regulatory protein (GKRP). During fasting, GKRP binds, inactivates and sequesters GK in the nucleus, which removes GK from the gluconeogenic process and prevents a futile cycle of glucose phosphorylation. Compounds that directly hyperactivate GK (GK activators) lower blood glucose levels and are being evaluated clinically as potential therapeutics for the treatment of type II diabetes mellitus. However, initial reports indicate that an increased risk of hypoglycaemia is associated with some GK activators. To mitigate the risk of hypoglycaemia, we sought to increase GK activity by blocking GKRP. Here we describe the identification of two potent small-molecule GK-GKRP disruptors (AMG-1694 and AMG-3969) that normalized blood glucose levels in several rodent models of diabetes. These compounds potently reversed the inhibitory effect of GKRP on GK activity and promoted GK translocation both in vitro (isolated hepatocytes) and in vivo (liver). A co-crystal structure of full-length human GKRP in complex with AMG-1694 revealed a previously unknown binding pocket in GKRP distinct from that of the phosphofructose-binding site. Furthermore, with AMG-1694 and AMG-3969 (but not GK activators), blood glucose lowering was restricted to diabetic and not normoglycaemic animals. These findings exploit a new cellular mechanism for lowering blood glucose levels with reduced potential for hypoglycaemic risk in patients with type II diabetes mellitus.
Asunto(s)
Proteínas Portadoras/antagonistas & inhibidores , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/farmacología , Hipoglucemiantes/uso terapéutico , Proteínas Adaptadoras Transductoras de Señales , Animales , Glucemia/metabolismo , Proteínas Portadoras/metabolismo , Núcleo Celular/enzimología , Cristalografía por Rayos X , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/enzimología , Modelos Animales de Enfermedad , Hepatocitos , Humanos , Hiperglucemia/sangre , Hiperglucemia/tratamiento farmacológico , Hiperglucemia/enzimología , Hipoglucemiantes/química , Hígado/citología , Hígado/enzimología , Hígado/metabolismo , Masculino , Modelos Moleculares , Especificidad de Órganos , Fosforilación/efectos de los fármacos , Piperazinas/química , Piperazinas/metabolismo , Piperazinas/farmacología , Piperazinas/uso terapéutico , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Ratas , Ratas Wistar , Sulfonamidas/química , Sulfonamidas/metabolismo , Sulfonamidas/farmacología , Sulfonamidas/uso terapéuticoRESUMEN
MicroRNAs (miRNAs) are a class of powerful posttranscriptional regulators implicated in the control of diverse biological processes, including regulation of hematopoiesis and the immune response. To define the biological functions of miR-142, which is preferentially and abundantly expressed in immune cells, we created a mouse line with a targeted deletion of this gene. Our analysis of miR-142(-/-) mice revealed a critical role for this miRNA in the development and homeostasis of lymphocytes. Marginal zone B cells expand in the knockout spleen, whereas the number of T and B1 B cells in the periphery is reduced. Abnormal development of hematopoietic lineages in miR-142(-/-) animals is accompanied by a profound immunodeficiency, manifested by hypoimmunoglobulinemia and failure to mount a productive immune response to soluble antigens and virus. miR-142(-/-) B cells express elevated levels of B-cell-activating factor (BAFF) receptor (BAFF-R) and as a result proliferate more robustly in response to BAFF stimulation. Lowering the BAFF-R gene dose in miR-142(-/-) mice rescues the B-cell expansion defect, suggesting that BAFF-R is a bona fide miR-142 target through which it controls B-cell homeostasis. Collectively, our results uncover miR-142 as an essential regulator of lymphopoiesis, and suggest that lesions in this miRNA gene may lead to primary immunodeficiency.
Asunto(s)
Linfocitos B/patología , Eliminación de Gen , Síndromes de Inmunodeficiencia/genética , Trastornos Inmunoproliferativos/genética , Linfopoyesis , MicroARNs/genética , Animales , Receptor del Factor Activador de Células B/genética , Linfocitos B/inmunología , Linfocitos B/metabolismo , Femenino , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Inmunidad Celular , Inmunidad Humoral , Síndromes de Inmunodeficiencia/inmunología , Síndromes de Inmunodeficiencia/patología , Trastornos Inmunoproliferativos/inmunología , Trastornos Inmunoproliferativos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/inmunologíaRESUMEN
The ß-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is one of the most hotly pursued targets for the treatment of Alzheimer's disease. We used a structure- and property-based drug design approach to identify 2-aminooxazoline 3-azaxanthenes as potent BACE1 inhibitors which significantly reduced CSF and brain Aß levels in a rat pharmacodynamic model. Compared to the initial lead 2, compound 28 exhibited reduced potential for QTc prolongation in a non-human primate cardiovascular safety model.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Xantenos/química , Xantenos/farmacología , Enfermedad de Alzheimer/tratamiento farmacológico , Animales , Línea Celular , Células HEK293 , Humanos , Inhibidores de Proteasas/síntesis química , Ratas , Xantenos/síntesis químicaRESUMEN
Wnt-modulator in surface ectoderm (WISE) is a secreted modulator of Wnt signaling expressed in the adult kidney. Activation of Wnt signaling has been observed in renal transplants developing interstitial fibrosis and tubular atrophy; however, whether WISE contributes to chronic changes is not well understood. Here, we found moderate to high expression of WISE mRNA in a rat model of renal transplantation and in kidneys from normal rats. Treatment with a neutralizing antibody against WISE improved proteinuria and graft function, which correlated with higher levels of ß-catenin protein in kidney allografts. In addition, treatment with the anti-WISE antibody reduced infiltration of CD68(+) macrophages and CD8(+) T cells, attenuated glomerular and interstitial injury, and decreased biomarkers of renal injury. This treatment reduced expression of genes involved in immune responses and in fibrogenic pathways. In summary, WISE contributes to renal dysfunction by promoting tubular atrophy and interstitial fibrosis.
Asunto(s)
Proteínas Portadoras/metabolismo , Trasplante de Riñón , Riñón/metabolismo , Insuficiencia Renal/prevención & control , Proteínas Wnt/metabolismo , Actinas/metabolismo , Animales , Anticuerpos/uso terapéutico , Biomarcadores/orina , Cadherinas/metabolismo , Proteínas Portadoras/antagonistas & inhibidores , Células Epiteliales/metabolismo , Fibroblastos/metabolismo , Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular , Riñón/inmunología , Pruebas de Función Renal , Masculino , Ratas , Ratas Endogámicas F344 , Ratas Endogámicas Lew , Insuficiencia Renal/orina , beta Catenina/metabolismoRESUMEN
FGF19 and FGF21 are distinctive members of the FGF family that function as endocrine hormones. Their potent effects on normalizing glucose, lipid, and energy homeostasis in disease models have made them an interesting focus of research for combating the growing epidemics of diabetes and obesity. Despite overlapping functions, FGF19 and FGF21 have many discrete effects, the most important being that FGF19 has both metabolic and proliferative effects, whereas FGF21 has only metabolic effects. Here we identify the structural determinants dictating differential receptor interactions that explain and distinguish these two physiological functions. We also have generated FGF19 variants that have lost the ability to induce hepatocyte proliferation but that still are effective in lowering plasma glucose levels and improving insulin sensitivity in mice. Our results add valuable insight into the structure-function relationship of FGF19/FGF21 and identify the structural basis underpinning the distinct proliferative feature of FGF19 compared with FGF21. In addition, these studies provide a road map for engineering FGF19 as a potential therapeutic candidate for treating diabetes and obesity.
Asunto(s)
Glucemia , Proliferación Celular , Factores de Crecimiento de Fibroblastos/fisiología , Hepatocitos/citología , Secuencia de Aminoácidos , Animales , Diabetes Mellitus/tratamiento farmacológico , Variación Genética , Resistencia a la Insulina , Ratones , Obesidad/tratamiento farmacológico , Fragmentos de Péptidos/farmacología , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes/farmacologíaRESUMEN
Aging of hematopoietic stem cells (HSCs) is linked to various blood disorders and malignancies. SIRT1 has been implicated in healthy aging, but its role in HSC aging is poorly understood. Surprisingly, we found that Sirt1 knockout improved the maintenance of quiescence of aging HSCs and their functionality as well as mouse survival in serial bone marrow transplantation (BMT) recipients. The majority of secondary and tertiary BMT recipients of aging wild type donor cells developed B/myeloid mixed phenotype acute leukemia (MPAL), which was markedly inhibited by Sirt1 knockout. SIRT1 inhibition also reduced the growth and survival of human B/myeloid MPAL cells. Sirt1 knockout suppressed global gene activation in old HSCs, prominently the genes regulating protein synthesis and oxidative metabolism, which may involve multiple downstream transcriptional factors. Our results demonstrate an unexpected role of SIRT1 in promoting HSC aging and age-dependent MPAL and suggest SIRT1 may be a new therapeutic target for modulating functions of aging HSCs and treatment of MPAL.
Asunto(s)
Leucemia , Sirtuina 1 , Envejecimiento/genética , Animales , Células Madre Hematopoyéticas/metabolismo , Leucemia/genética , Leucemia/metabolismo , Ratones , Fenotipo , Sirtuina 1/genética , Sirtuina 1/metabolismoRESUMEN
FGF19 and FGF21, unique members of the fibroblast growth factor (FGF) family, are hormones that regulate glucose, lipid, and energy homeostasis. Increased hepatocyte proliferation and liver tumor formation have also been observed in FGF19 transgenic mice. Here, we report that, in contrast to FGF19, FGF21 does not induce hepatocyte proliferation in vivo. To identify the mechanism for FGF19-induced hepatocyte proliferation, we explored similarities and differences in receptor specificity between FGF19 and FGF21. We find that although both are able to activate FGF receptors (FGFRs) 1c, 2c, and 3c, only FGF19 activates FGFR4, the predominant receptor in the liver. Using a C-terminal truncation mutant of FGF19 and a series of FGF19/FGF21 chimeric molecules, we determined that amino acids residues 38-42 of FGF19 are sufficient to confer both FGFR4 activation and increased hepatocyte proliferation in vivo to FGF21. These data suggest that activation of FGFR4 is the mechanism whereby FGF19 can increase hepatocyte proliferation and induce hepatocellular carcinoma formation.
Asunto(s)
Carcinoma Hepatocelular/mortalidad , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Hepatocitos/metabolismo , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Células 3T3-L1 , Secuencia de Aminoácidos , Animales , Carcinoma Hepatocelular/genética , Transformación Celular Neoplásica/genética , Femenino , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/farmacología , Humanos , Ratones , Ratones Transgénicos , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/genética , Eliminación de SecuenciaRESUMEN
Ewing's and osteogenic sarcoma are two of the leading causes of cancer deaths in children and adolescents. Recent data suggest that sarcomas may depend on the insulin-like growth factor type 1 (IGF-1) receptor (IGF1R) and/or the insulin receptor (INSR) to drive tumor growth, survival, and resistance to mammalian target of rapamycin complex 1 (mTORC1) inhibitors. We evaluated the therapeutic value of ganitumab (AMG 479; C(6472)H(10028)N(1728)O(2020)S(42)), an anti-IGF1R, fully human monoclonal antibody, alone and in combination with rapamycin (mTORC1 inhibitor) in Ewing's (SK-ES-1 and A673) and osteogenic (SJSA-1) sarcoma models. IGF1R was activated by IGF-1 but not by insulin in each sarcoma model. INSR was also activated by IGF-1 in the SJSA-1 and SK-ES-1 models, but not in the A673 model where insulin was the preferred INSR ligand. Ganitumab significantly inhibited the growth of SJSA-1 and SK-ES-1 xenografts; inhibition was associated with decreased IGF1R and Akt phosphorylation, reduced total IGF1R and bromodeoxyuridine detection, and increased caspase-3 expression. Ganitumab inhibited rapamycin-induced IGF1R, Akt, and glycogen synthase kinase-3ß hyperphosphorylation in each sarcoma model. However, ganitumab in combination with rapamycin also resulted in a marked increase in INSR expression and activity in the SJSA-1 and A673 models. The in vivo efficacy of ganitumab in the two ganitumab-sensitive models (SJSA-1 and SK-ES-1) was significantly enhanced in combination with rapamycin. Our results support studying ganitumab in combination with mTORC1 inhibitors for the treatment of sarcomas and suggest that INSR signaling is an important mechanism of resistance to IGF1R blockade.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Óseas/tratamiento farmacológico , Osteosarcoma/tratamiento farmacológico , Sarcoma de Ewing/tratamiento farmacológico , Sirolimus/uso terapéutico , Animales , Antibióticos Antineoplásicos/farmacología , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias Óseas/metabolismo , Caspasa 3/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Factor I del Crecimiento Similar a la Insulina/antagonistas & inhibidores , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones , Ratones Desnudos , Osteosarcoma/metabolismo , Fosforilación/efectos de los fármacos , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/antagonistas & inhibidores , Receptor de Insulina/metabolismo , Sarcoma de Ewing/metabolismo , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Seven novel and potent Raf small molecule kinase inhibitors (C1-7) were evaluated in seven-day oral repeat dose rat toxicity studies. All compounds tested induced hyperplasia in multiple tissues. Consistently affected was stratified squamous epithelium at a number of sites and transitional epithelium of urinary bladder and kidney. A seven-day time course study in rats showed morphologic evidence of epithelial proliferation in the nonglandular stomach within four to five hours after a single dose of C-1. Similar indications of cellular proliferation were observed in the urinary bladder by day 2 and in the heart, kidney, and liver by day 3. Transcriptional evidence of proliferation in the urinary bladder was detected within four to five hours after a single dose consistent with activation of the PI3K/AKT and ERK/MAPK pathways. In a twenty-eight-day rat toxicity study of C-1, hyperplasia was observed in the esophagus, nonglandular stomach, skin, urinary bladder, kidney, and heart. Hyperplasia of transitional epithelium of the urinary bladder was particularly severe and in one female rat was accompanied by the presence of a transitional cell carcinoma. These results suggest that these Raf inhibitors induce early transcriptional changes driving unchecked cell proliferation, resulting in marked tissue hyperplasia that can progress to carcinoma within a short time frame.
Asunto(s)
Inhibidores de Proteínas Quinasas/toxicidad , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Neoplasias de la Vejiga Urinaria/inducido químicamente , Neoplasias de la Vejiga Urinaria/enzimología , Animales , Proliferación Celular/efectos de los fármacos , Análisis por Conglomerados , Femenino , Histocitoquímica , Hiperplasia , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Mutación , Miocardio/patología , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Ratas , Ratas Sprague-Dawley , Nervio Ciático/efectos de los fármacos , Nervio Ciático/patología , Estómago/efectos de los fármacos , Estómago/patología , Distribución Tisular , Pruebas de Toxicidad Crónica , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Chronic myeloid leukemia (CML) is an age-dependent blood malignancy. Like many other age-dependent human diseases, laboratory animal research of CML uses young mice that do not factor in the influence of aging. To understand how aging may impact animal modeling of human age-dependent diseases, we established the first aging mouse model of human CML in BALB/c mice in the advanced age defined by 75% survival. This model was developed by noncytotoxic depletion of bone marrow lineage-positive cells followed by BCR-ABL retroviral transduction and transplantation. CML developed in aging mice shared many similarities to that in young mice, but had increased incidence of anemia that is often seen in human CML. Importantly, we showed that aging of both donor hematopoietic stem cells and recipient bone marrow niche impacted BCR-ABL mediated leukemogenesis and leukemia spectrum. Optimal CML induction relied on age-matching for donors and recipients, and cross-transplantation between young and old mice produced a mixture of different leukemia. Therefore, our model provides initial evidence of the feasibility and merit of CML modeling in aging mice and offers a new tool for future studies of CML stem cell drug resistance and therapeutic intervention in which aging would be taken into consideration as an influencing factor.
Asunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva , Animales , Células de la Médula Ósea , Proteínas de Fusión bcr-abl , Células Madre Hematopoyéticas , Ratones , Ratones Endogámicos BALB C , RetroviridaeRESUMEN
Uncontrolled hepatic glucose output (HGO) contributes significantly to the pathological hyperglycemic state of patients with type 2 diabetes. Glucagon, through action on its receptor, stimulates HGO, thereby leading to increased glycemia. Antagonizing the glucagon signaling pathway represents an attractive therapeutic approach for the treatment of type 2 diabetes. We previously reported the generation and characterization of several high-affinity monoclonal antibodies (mAbs) targeting the glucagon receptor (GCGR). In the present study, we demonstrate that a 5-week treatment of diet-induced obese mice with mAb effectively normalized nonfasting blood glucose. Similar treatment also reduced fasting blood glucose without inducing hypoglycemia or other undesirable metabolic perturbations. In addition, no hypoglycemia was found in db/db mice that were treated with a combination of insulin and mAb. Long-term treatment with the mAb caused dose-dependent hyperglucagonemia and minimal to mild alpha-cell hyperplasia in lean mice. There was no evidence of pancreatic alpha-cell neoplastic transformation in mice treated with mAb for as long as 18 weeks. Treatment-induced hyperglucagonemia and alpha-cell hyperplasia were reversible after treatment withdrawal for periods of 4 and 10 weeks, respectively. It is noteworthy that pancreatic beta-cell function was preserved, as demonstrated by improved glucose tolerance throughout the 18-week treatment period. Our studies further support the concept that long-term inhibition of GCGR signaling by a mAb could be an effective approach for controlling diabetic hyperglycemia.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Células Secretoras de Glucagón/patología , Glucagón/sangre , Glucosa/metabolismo , Hiperglucemia/tratamiento farmacológico , Hipoglucemiantes/uso terapéutico , Receptores de Glucagón/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/efectos adversos , Glucemia , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/metabolismo , Hiperglucemia/sangre , Hiperglucemia/metabolismo , Hiperplasia , Hipoglucemia/sangre , Hipoglucemia/metabolismo , Hipoglucemia/prevención & control , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/efectos adversos , Insulina/administración & dosificación , Insulina/uso terapéutico , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/sangre , Obesidad/metabolismo , TaquifilaxisRESUMEN
Sunitinib malate (SUTENT) is a multitargeted receptor tyrosine kinase (RTK) inhibitor that is approved multinationally for the treatment of imatinib-resistant/-intolerant gastrointestinal stromal tumor and advanced renal cell carcinoma. This paper characterizes the organ toxicity of sunitinib in Sprague-Dawley rats and cynomolgus monkeys, and the reversibility of any treatment-induced effects. Rats and monkeys received sunitinib (0-15 and 0-20 mg/kg/day, respectively) orally on a consecutive daily dosing schedule for thirteen weeks or on an intermittent daily dosing schedule for up to nine months. Clinical observations and laboratory parameters were recorded. Necropsy was conducted following treatment/recovery periods, and histologic examinations were performed. In rats, sunitinib was generally tolerated at 0.3 and 1.5 mg/kg/day, and findings were reversible. In monkeys, the level at which there were no observed adverse effects was 1.5 mg/kg/day, and findings were similarly reversible (except for uterine/ovarian weight changes and skin pallor). Data suggest that inhibition of multiple RTK pathways may induce pharmacologic effects on organ systems in nonclinical species. Key pharmacologic effects of sunitinib included reversible inhibition of neovascularization into the epiphyseal growth plate, and impaired corpora lutea formation and uterine development during estrus. Similar observations have been noted with this class of RTK signaling inhibitors and are consistent with pharmacologic perturbations of physiologic/angiogenic processes associated with the intended molecular targets.
Asunto(s)
Antineoplásicos/toxicidad , Indoles/toxicidad , Pirroles/toxicidad , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Pruebas de Toxicidad Crónica/métodos , Glándulas Suprarrenales/efectos de los fármacos , Glándulas Suprarrenales/patología , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Médula Ósea/efectos de los fármacos , Médula Ósea/patología , Femenino , Tracto Gastrointestinal/efectos de los fármacos , Tracto Gastrointestinal/patología , Placa de Crecimiento/efectos de los fármacos , Placa de Crecimiento/patología , Incisivo/efectos de los fármacos , Incisivo/patología , Indoles/administración & dosificación , Indoles/farmacocinética , Tejido Linfoide/efectos de los fármacos , Tejido Linfoide/patología , Macaca fascicularis , Masculino , Microscopía Electrónica , Neovascularización Patológica/inducido químicamente , Neovascularización Patológica/patología , Ovario/efectos de los fármacos , Ovario/patología , Páncreas/efectos de los fármacos , Páncreas/patología , Pirroles/administración & dosificación , Pirroles/farmacocinética , Ratas , Ratas Sprague-Dawley , Recuperación de la Función/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , SunitinibRESUMEN
Many patients with chronic inflammation of the gut, such as that observed in inflammatory bowel disease (IBD), develop colorectal cancer (CRC). Recent studies have reported that the development of IBD and CRC partly results from an imbalanced composition of intestinal microbiota and that intestinal inflammation in these diseases can be modulated by the microbiota. The human commensal Bacteroides fragilis is best exemplified playing a protective role against the development of experimental colitis in several animal disease models. In this study, we found that gut inflammation caused by dextran sulfate sodium (DSS) treatment was inhibited by B. fragilis colonization in mice. Further, we reveal a protective role of B. fragilis treatment against colon tumorigenesis using an azoxymethane (AOM)/DSS-induced model of colitis-associated colon cancer in mice and demonstrate that the decreased tumorigenesis by B. fragilis administration is accompanied by inhibited expression of C-C chemokine receptor 5 (CCR5) in the gut. We show direct evidence that the inhibition of tumor formation provided by B. fragilis in colitis-associated CRC animals was dependent on the production of polysaccharide A (PSA) from B. fragilis and that Toll-like receptor 2 (TLR2) signaling was responsible for the protective function of B. fragilisIMPORTANCE The incidence of colorectal cancer (CRC) is rapidly growing worldwide, and there is therefore a greater emphasis on studies of the treatment or prevention of CRC pathogenesis. Recent studies suggested that consideration of the microbiota is unavoidable to understand inflammation and tumorigenesis in the gastrointestinal tract. We demonstrate, using a mouse model of colitis-associated CRC, that human commensal B. fragilis protects against colon tumorigenesis. The protective role against tumor formation provided by B. fragilis is associated with inhibition of expression of the chemokine receptor CCR5 in the colon. The molecular mechanism for protection against CRC provided by B. fragilis is dependent on polysaccharide A production and is mediated by TLR2 signaling. Our results suggest that the commensal microorganism B. fragilis can be used to prevent inflammation-associated CRC development and may provide an effective therapeutic strategy for CRC.
Asunto(s)
Bacteroides fragilis/crecimiento & desarrollo , Colitis/complicaciones , Colitis/prevención & control , Neoplasias del Colon/prevención & control , Animales , Azoximetano/administración & dosificación , Colitis/inducido químicamente , Neoplasias del Colon/inducido químicamente , Sulfato de Dextran/administración & dosificación , Modelos Animales de Enfermedad , Ratones , Receptores CCR5/análisisRESUMEN
PURPOSE: Proliferating cell nuclear antigen (PCNA) plays an essential role in regulating DNA synthesis and repair and is indispensable to cancer cell growth and survival. We previously reported a novel cancer associated PCNA isoform (dubbed caPCNA), which was ubiquitously expressed in a broad range of cancer cells and tumor tissues, but not significantly in nonmalignant cells. We found the L126-Y133 region of caPCNA is structurally altered and more accessible to protein-protein interaction. A cell-permeable peptide harboring the L126-Y133 sequence blocked PCNA interaction in cancer cells and selectively kills cancer cells and xenograft tumors. On the basis of these findings, we sought small molecules targeting this peptide region as potential broad-spectrum anticancer agents. EXPERIMENTAL DESIGN: By computer modeling and medicinal chemistry targeting a surface pocket partly delineated by the L126-Y133 region of PCNA, we identified a potent PCNA inhibitor (AOH1160) and characterized its therapeutic properties and potential toxicity. RESULTS: AOH1160 selectively kills many types of cancer cells at below micromolar concentrations without causing significant toxicity to a broad range of nonmalignant cells. Mechanistically, AOH1160 interferes with DNA replication, blocks homologous recombination-mediated DNA repair, and causes cell-cycle arrest. It induces apoptosis in cancer cells and sensitizes them to cisplatin treatment. AOH1160 is orally available to animals and suppresses tumor growth in a dosage form compatible to clinical applications. Importantly, it does not cause significant toxicity at 2.5 times of an effective dose. CONCLUSIONS: These results demonstrated the favorable therapeutic properties and the potential of AOH1160 as a broad-spectrum therapeutic agent for cancer treatment.
Asunto(s)
Antineoplásicos/farmacología , Biomarcadores de Tumor , Antígeno Nuclear de Célula en Proliferación/metabolismo , Animales , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Roturas del ADN/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Desarrollo de Medicamentos , Humanos , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Antígeno Nuclear de Célula en Proliferación/química , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
AIM: To identify neuron-selective androgen receptor (AR) signaling inhibitors, which could be useful in the treatment of spinal and bulbar muscular atrophy (SBMA), or Kennedy's disease, a neuromuscular disorder in which deterioration of motor neurons leads to progressive muscle weakness. METHODS: Cell lines representing prostate, kidney, neuron, adipose, and muscle tissue were developed that stably expressed the CFP-AR-YFP FRET reporter. We used these cells to screen a library of small molecules for cell type-selective AR inhibitors. Secondary screening in luciferase assays was used to identify the best cell-type specific AR inhibitors. The mechanism of action of a neuron-selective AR inhibitor was examined in vitro using luciferase reporter assays, immunofluorescence microscopy, and immunoprecipitations. Rats were treated with the most potent compound and tissue-selective AR inhibition was examined using RT-qPCR of AR-regulated genes and immunohistochemistry. RESULTS: We identified the thiazole class of antibiotics as compounds able to inhibit AR signaling in a neuronal cell line but not a muscle cell line. One of these antibiotics, thiostrepton is able to inhibit the activity of both wild type and polyglutamine expanded AR in neuronal GT1-7 cells with nanomolar potency. The thiazole antibiotics are known to inhibit FOXM1 activity and accordingly, a novel FOXM1 inhibitor FDI-6 also inhibited AR activity in a neuron-selective fashion. The selective inhibition of AR is likely indirect as the varied structures of these compounds would not suggest that they are competitive antagonists. Indeed, we found that FOXM1 expression correlates with cell-type selectivity, FOXM1 co-localizes with AR in the nucleus, and that shRNA-mediated knock down of FOXM1 reduces AR activity and thiostrepton sensitivity in a neuronal cell line. Thiostrepton treatment reduces FOXM1 levels and the nuclear localization of beta-catenin, a known co-activator of both FOXM1 and AR, and reduces the association between beta-catenin and AR. Treatment of rats with thiostrepton demonstrated AR signaling inhibition in neurons, but not muscles. CONCLUSION: Our results suggest that thiazole antibiotics, or other inhibitors of the AR-FOXM1 axis, can inhibit AR signaling selectively in motor neurons and may be useful in the treatment or prevention of SBMA symptoms.
RESUMEN
The tumor suppressor gene RASSF1A is epigenetically silenced in most human cancers. As a binding partner of the kinases MST1 and MST2, the mammalian orthologs of the Drosophila Hippo kinase, RASSF1A is a potential regulator of the Hippo tumor suppressor pathway. RASSF1A shares these properties with the scaffold protein SAV1. The role of this pathway in human cancer has remained enigmatic inasmuch as Hippo pathway components are rarely mutated in tumors. Here we show that Rassf1a homozygous knockout mice develop liver tumors. However, heterozygous deletion of Sav1 or codeletion of Rassf1a and Sav1 produced liver tumors with much higher efficiency than single deletion of Rassf1a. Analysis of RASSF1A-binding partners by mass spectrometry identified the Hippo kinases MST1, MST2, and the oncogenic IκB kinase TBK1 as the most enriched RASSF1A-interacting proteins. The transcriptome of Rassf1a(-/-) livers was more deregulated than that of Sav1(+/-) livers, and the transcriptome of Rassf1a(-/-), Sav1(+/-) livers was similar to that of Rassf1a(-/-) mice. We found that the levels of TBK1 protein were substantially upregulated in livers lacking Rassf1a. Furthermore, transcripts of several ß-tubulin isoforms were increased in the Rassf1a-deficient livers presumably reflecting a role of RASSF1A as a microtubule-stabilizing protein. In human liver cancer, RASSF1A frequently undergoes methylation at the promoter but this was not observed for MST1, MST2, or SAV1. Our results suggest a multifactorial role of RASSF1A in suppression of liver carcinogenesis. Cancer Res; 76(9); 2824-35. ©2016 AACR.
Asunto(s)
Proteínas de Ciclo Celular/genética , Transformación Celular Neoplásica/genética , Neoplasias Hepáticas/genética , Proteínas Supresoras de Tumor/genética , Adenoma de Células Hepáticas/genética , Adenoma de Células Hepáticas/patología , Animales , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Proteínas de Ciclo Celular/metabolismo , Modelos Animales de Enfermedad , Inmunoprecipitación , Neoplasias Hepáticas/patología , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Even when treated with aggressive current therapies, most patients with glioblastoma survive less than two years. Rapid tumor growth, an invasive nature, and the blood-brain barrier, which limits the penetration of large molecules into the brain, all contribute to the poor tumor response associated with conventional therapies. Immunotherapy has emerged as a therapeutic approach that may overcome these challenges. We recently reported that single-walled carbon nanotubes (SWCNTs) can be used to dramatically increase the immunotherapeutic efficacy of CpG oligonucleotides in a mouse model of glioma. Following implantation in the mouse brain, the tumor cell line used in these previous studies (GL261) tends to form a spherical tumor with limited invasion into healthy brain. In order to evaluate SWCNT/CpG therapy under more clinically-relevant conditions, here we report the treatment of a more invasive mouse glioma model (K-Luc) that better recapitulates human disease. In addition, a CpG sequence previously tested in humans was used to formulate the SWCNT/CpG which was combined with temozolomide, the standard of care chemotherapy for glioblastoma patients. We found that, following two intracranial administrations, SWCNT/CpG is well-tolerated and improves the survival of mice bearing invasive gliomas. Interestingly, the efficacy of SWCNT/CpG was enhanced when combined with temozolomide. This enhanced anti-tumor efficacy was correlated to an increase of tumor-specific cytotoxic activity in splenocytes. These results reinforce the emerging understanding that immunotherapy can be enhanced by combining it with chemotherapy and support the continued development of SWCNT/CpG.