RESUMO
The purpose of this work was to use various molecular imaging techniques to non-invasively assess GSK2849330 (anti HER3 ADCC and CDC enhanced 'AccretaMab' monoclonal antibody) pharmacokinetics and pharmacodynamics in human xenograft tumor-bearing mice. Immuno-PET biodistribution imaging of radiolabeled 89Zr-GSK2849330 was assessed in mice with HER3 negative (MIA-PaCa-2) and positive (CHL-1) human xenograft tumors. Dose dependency of GSK2849330 disposition was assessed using varying doses of unlabeled GSK2849330 co-injected with 89Zr-GSK2849330. In-vivo NIRF optical imaging and ex-vivo confocal microscopy were used to assess the biodistribution of GSK2849330 and the HER3 receptor occupancy in HER3 positive xenograft tumors (BxPC3, and CHL-1). Ferumoxytol (USPIO) contrast-enhanced MRI was used to investigate the effects of GSK2849330 on tumor macrophage content in CHL-1 xenograft bearing mice. Immuno-PET imaging was used to monitor the whole body drug biodistribution and CHL-1 xenograft tumor uptake up to 144 hours post injection of 89Zr-GSK2849330. Both hepatic and tumor uptake were dose dependent and saturable. The optical imaging data in the BxPC3 xenograft tumor confirmed the tumor dose response finding in the Immuno-PET study. Confocal microscopy showed a distinguished cytoplasmic punctate staining pattern within individual CHL-1 cells. GSK2849330 inhibited tumor growth and this was associated with a significant decrease in MRI signal to noise ratio after USPIO injection and with a significant increase in tumor macrophages as confirmed by a quantitative immunohistochemistry analysis. By providing both dose response and time course data from both 89Zr and fluorescently labeled GSK2849330, complementary imaging studies were used to characterize GSK2849330 biodistribution and tumor uptake in vivo. Ferumoxytol-enhanced MRI was used to monitor aspects of the immune system response to GSK2849330. Together these approaches potentially provide clinically translatable, non-invasive techniques to support dose optimization, and assess immune activation and anti-tumor responses.
Assuntos
Anticorpos Monoclonais Humanizados/farmacocinética , Anticorpos Monoclonais/farmacocinética , Macrófagos/imunologia , Compostos Radiofarmacêuticos/farmacocinética , Receptor ErbB-3/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/uso terapêutico , Linhagem Celular Tumoral , Feminino , Óxido Ferroso-Férrico/química , Humanos , Imuno-Histoquímica , Marcação por Isótopo , Macrófagos/citologia , Macrófagos/patologia , Camundongos , Camundongos Nus , Neoplasias/diagnóstico por imagem , Neoplasias/tratamento farmacológico , Radioisótopos , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/uso terapêutico , Receptor ErbB-3/metabolismo , Distribuição Tecidual , Transplante Heterólogo , Zircônio/químicaRESUMO
UNLABELLED: Aberrant WNT signaling is associated with the formation and growth of numerous human cancer types. The low-density lipoprotein receptor-related protein 6 (LRP6) is the least redundant component of the WNT receptor complex with two independent WNT ligand-binding sites. Using domain antibody (dAb) technology, a bispecific antibody (GSK3178022) to LRP6 was identified that is capable of blocking stimulation in the presence of a range of WNT and R-spondin (RSPO) ligands in vitro GSK3178022 was also efficacious in reducing WNT target gene expression in vivo, in both cancer cell line and patient-derived xenograft models, and delays tumor growth in a patient-derived RSPO fusion model of colorectal cancer. IMPLICATIONS: This article demonstrates the inhibition of a key oncogenic receptor, intractable to mAb inhibition due to multiple independent ligand interaction sites, using an innovative dAb approach. Mol Cancer Res; 14(9); 859-68. ©2016 AACR.
Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/imunologia , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Anticorpos Biespecíficos/farmacocinética , Linhagem Celular Tumoral , Feminino , Fibrossarcoma/imunologia , Fibrossarcoma/metabolismo , Fibrossarcoma/patologia , Fibrossarcoma/terapia , Células HEK293 , Humanos , Ligantes , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/antagonistas & inibidores , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Trombospondinas/antagonistas & inibidores , Trombospondinas/imunologia , Trombospondinas/metabolismo , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/imunologia , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
B-cell maturation antigen (BCMA, also termed TNFRSF17) is an attractive therapeutic target due to its restricted expression on normal and malignant plasma cells (PC). GSK2857916 (or J6M0-MMAF) is a BCMA-specific antibody conjugated to the microtubule-disrupting agent monomethyl auristatin F (MMAF) via a protease-resistant linker. To evaluate the clinical potential of this agent, tumour cells from seventy multiple myeloma (MM) patients were assessed for BCMA expression by immunohistochemistry and flow cytometry. All patients tested expressed BCMA, at varying levels, and both surface and intracellular expression were observed. BCMA expression is maintained through relapse, extramedullary spread and in residual disease post therapy. BCMA levels may also be prognostically useful as higher levels of BCMA were associated with poorer outcomes, even taking into account genetic risk. We observed rapid internalization of surface BCMA and newly expressed protein by 1 h, suggesting a mechanism for J6M0-MMAF activity even with low surface antigen. J6M0-MMAF mediated cytotoxicity of MM cells varied with dose and antigen levels, with clonogenic progenitors killed at lower doses than mature cells. In comparison, J6M0-MMAF killing of primary CD138(+) myeloma cells occurred with slower kinetics. Our observations support BCMA to be a promising therapeutic target in MM for novel therapies such as J6M0-MMAF.
Assuntos
Antígeno de Maturação de Linfócitos B/antagonistas & inibidores , Imunoconjugados/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antígeno de Maturação de Linfócitos B/genética , Antígeno de Maturação de Linfócitos B/metabolismo , Medula Óssea/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citometria de Fluxo , Seguimentos , Expressão Gênica , Humanos , Imunoconjugados/farmacologia , Imuno-Histoquímica , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/mortalidade , Plasmócitos/metabolismo , Plasmócitos/patologia , PrognósticoRESUMO
Fibroblast growth factor (FGF) ligand-dependent signaling has a fundamental role in cancer development and tumor maintenance. GSK3052230 (also known as FP-1039) is a soluble decoy receptor that sequesters FGFs and inhibits FGFR signaling. Herein, the efficacy of this molecule was tested in models of mesothelioma, a tumor type shown to express high levels of FGF2 and FGFR1. GSK3052230 demonstrated antiproliferative activity across a panel of mesothelioma cell lines and inhibited growth of tumor xenografts in mice. High expression of FGF2 and FGFR1 correlated well with response to FGF pathway inhibition. GSK3052230 inhibited MAPK signaling as evidenced by decreased phospho-ERK and phospho-S6 levels in vitro and in vivo. Additionally, dose-dependent and statistically-significant reductions in tumor vessel density were observed in GSK3052230-treated tumors compared to vehicle-treated tumors. These data support the role of GSK3052230 in effectively targeting FGF-FGFR autocrine signaling in mesothelioma, demonstrate its impact on tumor growth and angiogenesis, and provide a rationale for the current clinical evaluation of this molecule in mesothelioma patients.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Mesotelioma/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Animais , Comunicação Autócrina , Linhagem Celular , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoglobulina G/química , Ligantes , Imageamento por Ressonância Magnética , Camundongos , Camundongos SCID , Transplante de Neoplasias , Neovascularização Patológica , Proteínas de Fusão Oncogênica/química , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/química , Proteínas Recombinantes de FusãoRESUMO
B-cell maturation antigen (BCMA), highly expressed on malignant plasma cells in human multiple myeloma (MM), has not been effectively targeted with therapeutic monoclonal antibodies. We here show that BCMA is universally expressed on the MM cell surface and determine specific anti-MM activity of J6M0-mcMMAF (GSK2857916), a novel humanized and afucosylated antagonistic anti-BCMA antibody-drug conjugate via a noncleavable linker. J6M0-mcMMAF specifically blocks cell growth via G2/M arrest and induces caspase 3-dependent apoptosis in MM cells, alone and in coculture with bone marrow stromal cells or various effector cells. It strongly inhibits colony formation by MM cells while sparing surrounding BCMA-negative normal cells. J6M0-mcMMAF significantly induces effector cell-mediated lysis against allogeneic or autologous patient MM cells, with increased potency and efficacy compared with the wild-type J6M0 without Fc enhancement. The antibody-dependent cell-mediated cytotoxicity and apoptotic activity of J6M0-mcMMAF is further enhanced by lenalidomide. Importantly, J6M0-mcMMAF rapidly eliminates myeloma cells in subcutaneous and disseminated mouse models, and mice remain tumor-free up to 3.5 months. Furthermore, J6M0-mcMMAF recruits macrophages and mediates antibody-dependent cellular phagocytosis of MM cells. Together, these results demonstrate that GSK2857916 has potent and selective anti-MM activities via multiple cytotoxic mechanisms, providing a promising next-generation immunotherapeutic in this cancer.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígeno de Maturação de Linfócitos B/imunologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/patologia , Imunotoxinas/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Linfócitos B/imunologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Fatores Imunológicos/imunologia , Fatores Imunológicos/uso terapêutico , Imunotoxinas/imunologia , Lenalidomida , Camundongos , Camundongos SCID , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Talidomida/análogos & derivados , Talidomida/imunologia , Talidomida/uso terapêuticoRESUMO
Thymosin beta 4 (Tß4) was previously shown to reduce infarct size and improve contractile performance in chronic myocardial ischemic injury via two phases of action: an acute phase, just after injury, when Tß4 preserves ischemic myocardium via antiapoptotic or anti-inflammatory mechanisms; and a chronic phase, when Tß4 activates the growth of vascular or cardiac progenitor cells. In order to differentiate between the effects of Tß4 during the acute and during the chronic phases, and also in order to obtain detailed hemodynamic and biomarker data on the effects of Tß4 treatment suitable for use in clinical studies, we tested Tß4 in a rat model of chronic myocardial ischemia using two dosing regimens: short term dosing (Tß4 administered only during the first 3 days following injury), and long term dosing (Tß4 administered during the first 3 days following injury and also every third day until the end of the study). Tß4 administered throughout the study reduced infarct size and resulted in significant improvements in hemodynamic performance; however, chamber volumes and ejection fractions were not significantly improved. Tß4 administered only during the first 3 days following injury tended to reduce infarct size, chamber volumes and improve hemodynamic performance. Plasma biomarkers of myocyte injury were significantly reduced by Tß4 treatment during the acute injury period, and plasma ANP levels were significantly reduced in both dosing groups. Surprisingly, neither acute nor chronic Tß4 treatment significantly increased blood vessel density in peri-infarct regions. These results suggest the following: repeated dosing may be required to achieve clinically measureable improvements in cardiac function post-myocardial infarction (MI); improvement in cardiac function may be observed in the absence of a high degree of angiogenesis; and that plasma biomarkers of cardiac function and myocardial injury are sensitive pharmacodynamic biomarkers of the effects of Tß4.
RESUMO
Epoxyeicosatrienoic acids, substrates for soluble epoxide hydrolase (sEH), exhibit vasodilatory and antihypertrophic activities. Inhibitors of sEH might therefore hold promise as heart failure therapeutics. We examined the ability of sEH inhibitors GSK2188931 and GSK2256294 to modulate cardiac hypertrophy, fibrosis, and function after transverse aortic constriction (TAC) in rats and mice. GSK2188931 administration was initiated in rats 1 day before TAC, whereas GSK2256294 treatment was initiated in mice 2 weeks after TAC. Four weeks later, cardiovascular function was assessed, plasma was collected for drug and sEH biomarker concentrations, and left ventricle was isolated for messenger RNA and histological analyses. In rats, although GSK2188931 prevented TAC-mediated increases in certain genes associated with hypertrophy and fibrosis (α-skeletal actin and connective tissue growth factor), the compound failed to attenuate TAC-induced increases in left ventricle mass, posterior wall thickness, end-diastolic volume and pressure, and perivascular fibrosis. Similarly, in mice, GSK2256294 did not reverse cardiac remodeling or systolic dysfunction induced by TAC. Both compounds increased the sEH substrate/product (leukotoxin/leukotoxin diol) ratio, indicating sEH inhibition. In summary, sEH inhibition does not prevent cardiac remodeling or dysfunction after TAC. Thus, targeting sEH seems to be insufficient for reducing pressure overload hypertrophy.
Assuntos
Aorta/efeitos dos fármacos , Cicloexilaminas/farmacologia , Inibidores Enzimáticos/farmacologia , Epóxido Hidrolases/antagonistas & inibidores , Piperidinas/farmacologia , Triazinas/farmacologia , Animais , Aorta/patologia , Cardiomegalia/tratamento farmacológico , Cardiomegalia/patologia , Constrição Patológica , Modelos Animais de Doenças , Fibrose , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Remodelação Ventricular/efeitos dos fármacosRESUMO
Pulmonary edema resulting from high pulmonary venous pressure (PVP) is a major cause of morbidity and mortality in heart failure (HF) patients, but current treatment options demonstrate substantial limitations. Recent evidence from rodent lungs suggests that PVP-induced edema is driven by activation of pulmonary capillary endothelial transient receptor potential vanilloid 4 (TRPV4) channels. To examine the therapeutic potential of this mechanism, we evaluated TRPV4 expression in human congestive HF lungs and developed small-molecule TRPV4 channel blockers for testing in animal models of HF. TRPV4 immunolabeling of human lung sections demonstrated expression of TRPV4 in the pulmonary vasculature that was enhanced in sections from HF patients compared to controls. GSK2193874 was identified as a selective, orally active TRPV4 blocker that inhibits Ca(2+) influx through recombinant TRPV4 channels and native endothelial TRPV4 currents. In isolated rodent and canine lungs, TRPV4 blockade prevented the increased vascular permeability and resultant pulmonary edema associated with elevated PVP. Furthermore, in both acute and chronic HF models, GSK2193874 pretreatment inhibited the formation of pulmonary edema and enhanced arterial oxygenation. Finally, GSK2193874 treatment resolved pulmonary edema already established by myocardial infarction in mice. These findings identify a crucial role for TRPV4 in the formation of HF-induced pulmonary edema and suggest that TRPV4 blockade is a potential therapeutic strategy for HF patients.
Assuntos
Insuficiência Cardíaca/complicações , Moduladores de Transporte de Membrana/administração & dosagem , Moduladores de Transporte de Membrana/uso terapêutico , Edema Pulmonar/tratamento farmacológico , Edema Pulmonar/prevenção & controle , Canais de Cátion TRPV/antagonistas & inibidores , Administração Oral , Animais , Pressão Sanguínea/efeitos dos fármacos , Cálcio/metabolismo , Modelos Animais de Doenças , Diuréticos/farmacologia , Endotélio/efeitos dos fármacos , Endotélio/metabolismo , Endotélio/patologia , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Frequência Cardíaca/efeitos dos fármacos , Humanos , Técnicas In Vitro , Ativação do Canal Iônico/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Moduladores de Transporte de Membrana/química , Moduladores de Transporte de Membrana/farmacologia , Camundongos , Camundongos Knockout , Permeabilidade/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Edema Pulmonar/etiologia , Edema Pulmonar/patologia , Ratos , Canais de Cátion TRPV/metabolismo , Equilíbrio Hidroeletrolítico/efeitos dos fármacosRESUMO
PURPOSE: To test whether B-22956/1, a novel intravascular contrast agent with a high affinity to serum albumin (Bracco Imaging SpA.), allowed quantifying neovessel and macrophage density in atherosclerotic plaques of rabbits using MRI. MATERIALS AND METHODS: A T1-weighted MRI of the aorta was acquired in 10 rabbits (7 atherosclerotic and 3 control rabbits) before and up to 2 h after intravenous injection of 100 mumol/kg of Gd-DTPA or 75 mumol/kg of B-22956/1. Plaque enhancement was measured at different time points. Immunohistochemistry was performed using anti-CD 31 antibodies and anti-RAM 11 antibodies to correlate to neovessel and macrophage density, respectively. RESULTS: MRI showed a significant plaque enhancement 2 h after B-22956/1 versus Gd-DTPA in the atherosclerotic group (39.75% versus 9.5%; P < 0.0001. Early atherosclerotic plaques (n = 146) enhancement positively correlates with neovessel density on corresponding histological sections (r = 0.42; P < 0.01). Enhancement of atherosclerotic plaques 2 h after injection of B-22956/1 correlated with macrophage density (r = 0.71; P < 0.01). CONCLUSION: Enhancement of atherosclerotic plaques with MRI correlated with neovessel density at early time points after the injection of B-22956/1 and with macrophage density, at later time points. Hence, B-22956/1-enhanced MRI represents a promising imaging technique for the identification of "high-risk" plaques.
Assuntos
Albuminas/metabolismo , Aterosclerose/diagnóstico , Meios de Contraste/administração & dosagem , Imageamento por Ressonância Magnética/métodos , Neovascularização Patológica/diagnóstico , Compostos Organometálicos , Animais , Aorta Abdominal/patologia , Aorta Abdominal/ultraestrutura , Aterosclerose/patologia , Modelos Animais de Doenças , Gadolínio DTPA , Aumento da Imagem/métodos , Processamento de Imagem Assistida por Computador/métodos , Masculino , Neovascularização Patológica/patologia , Coelhos , Fatores de TempoRESUMO
OBJECTIVE: Osteoporosis trials suggest raloxifene decreased cardiovascular events in women with pre-existing atherosclerosis. We assessed the hypothesis that selective estrogen receptor modulation induces plaque stability in "menopausal" animals. METHODS AND RESULTS: Atherosclerosis was induced in 42 ovariectomized New Zealand white rabbits by cholesterol feeding and mechanical injury. Animals were imaged by magnetic resonance imaging (MRI) for baseline atherosclerosis, and randomized to control (OVX (ovariectomized control group), n=12), raloxifene 35-60 mg/kg/day by diet admixture (RLX (raloxifene therapy group), n=24), or immediate sacrifice (n=6) for immunohistopathologic correlation of MRI. Six months later, rabbits underwent repeat MRI then sacrifice for micro-computed tomography (microCT) and molecular analysis. Unlike OVX, RLX reduced atheroma volume. Analysis for lesion inflammation revealed reductions in COX-2 (cyclooxygenase-2), MMP-1 (matrix metalloproteinase-1), MCP-1 (monocyte chemoattractant protein-1) expression and macrophage infiltration in RLX versus OVX with concomitant upregulation of estrogen receptor alpha (ERalpha). microCT showed similar total vascular calcification between groups, but calcifications in RLX were less nodular with better radial organization (mean calcific arc angle 63+/-7 degrees versus 33+/-6 degrees in OVX), the predicted result of a 53% increase in BMP-2 (bone-morphogenetic protein-2). CONCLUSIONS: Raloxifene treatment results in reduced lesion volume, enhanced mechanical stability of vascular calcification, and less inflamed lesions characterized by less macrophage infiltration and reduced COX-2, MMP-1 and MCP-1 expression.
Assuntos
Aterosclerose/tratamento farmacológico , Aterosclerose/patologia , Menopausa , Osteoporose/tratamento farmacológico , Cloridrato de Raloxifeno/uso terapêutico , Moduladores Seletivos de Receptor Estrogênico/uso terapêutico , Animais , Aterosclerose/metabolismo , Quimiocina CCL2/metabolismo , Ciclo-Oxigenase 2/metabolismo , Modelos Animais de Doenças , Feminino , Imageamento por Ressonância Magnética , Metaloproteinase 1 da Matriz/metabolismo , Osteoporose/metabolismo , Osteoporose/patologia , Ovariectomia , CoelhosRESUMO
The inflammatory response is critical to the development and progression of heart failure. Chemokines and their receptors are a distinct class of inflammatory modulators that may play a role in mediating myocardial dysfunction in heart failure. Levels of the chemokine CXCL12, also known as stromal cell-derived factor (SDF), and its receptor, CXCR4, are elevated in patients with heart failure, and we undertook this study to determine whether this chemokine system can directly affect cardiac function in the absence of leukocytes. Murine papillary muscles and adult rat cardiac myocytes treated with CXCL12, the only identified ligand of CXCR4, demonstrate blunted inotropic responses to physiologic concentrations of calcium. The negative inotropic effects on cardiac myocytes are accompanied by a proportional diminution of calcium transients. The effects are abrogated by AMD3100, a specific CXCR4 inhibitor. Overexpression of the receptor through adenoviral infection with a CXCR4 construct accentuates the negative inotropic effects of CXCL12 on cardiac myocytes during calcium stimulation. CXCR4 activation also attenuates beta-adrenergic-mediated increases in calcium mobilization and fractional shortening in cardiac myocytes. In electrophysiologic studies, CXCL12 decreases forskolin- and isoproterenol-induced voltage-gated L-type calcium channel activation. These studies demonstrate that activation of CXCR4 results in a direct negative inotropic modulation of cardiac myocyte function. The specific mechanism of action involves alterations of calcium channel activity on the membrane. The presence of functional CXCR4 on cardiac myocytes introduces a new target for treating cardiac dysfunction.
Assuntos
Contração Miocárdica , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Receptores CXCR4/metabolismo , Adenoviridae/genética , Animais , Cálcio/metabolismo , Cálcio/farmacologia , Canais de Cálcio/metabolismo , Quimiocina CXCL12 , Quimiocinas CXC/farmacologia , AMP Cíclico/farmacologia , Sinergismo Farmacológico , Vetores Genéticos , Ventrículos do Coração/metabolismo , Técnicas In Vitro , Isoproterenol/farmacologia , Masculino , Camundongos , Músculos Papilares/fisiologia , Ratos , Receptores CXCR4/fisiologiaRESUMO
Hyperlipidemia promotes oxidant stress, inflammation, and atherogenesis in apolipoprotein E-deficient (ApoE((-/-))) mice. Mice transgenic for lysozyme (LZ-Tg) are resistant to acute and chronic oxidative stress and have decreased circulating levels of pro-oxidant advanced glycation end-products (AGEs). Herein we report that TIB-186 macrophages transduced with adenovirus-expressing human LZ (AdV-LZ) containing the AGE-binding domain facilitated AGE uptake and degradation and that AdV-LZ-transduced macrophages and peritoneal macrophages from LZ-Tg mice suppressed the AGE-triggered tumor necrosis factor-alpha response. We assessed atherosclerosis in LZ-Tg mice crossed with ApoE((-/-)) mice (LZ/ApoE((-/-))) and found increased serum LZ levels and decreased AGE and 8-isoprostanes levels, although hyperlipidemia remained similar to ApoE((-/-)) controls. Atherosclerotic plaques and neointimal lesions at the aortic root and descending aorta were markedly decreased (by 40% and 80%, respectively) in LZ/ApoE((-/-)) versus ApoE((-/-)) mice, as were inflammatory infiltrates. The arterial lesions following femoral artery injury in LZ/ApoE((-/-)) mice were suppressed (intimal to media ratio decreased by 50%), as were AGE deposits and vascular smooth muscle cell activation, compared to ApoE((-/-)) mice. Despite hyperlipidemia, development of atheroma and occlusive, inflammatory arterial neointimal lesions in response to injury was suppressed in LZ/ApoE((-/-)) mice. This effect may be due to the antioxidant properties of LZ, which is possibly linked to the AGE-binding domain region of the molecule.
Assuntos
Aterosclerose/patologia , Produtos Finais de Glicação Avançada/metabolismo , Hiperlipidemias/complicações , Muramidase/genética , Muramidase/metabolismo , Adenoviridae/genética , Animais , Aorta/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/enzimologia , Aterosclerose/etiologia , Aterosclerose/metabolismo , Western Blotting , Células Cultivadas , Artéria Femoral/lesões , Artéria Femoral/patologia , Vetores Genéticos , Humanos , Imuno-Histoquímica , Macrófagos/metabolismo , Camundongos , Camundongos Transgênicos , Músculo Liso Vascular/metabolismo , RNA Mensageiro/análise , Proteínas Recombinantes , Transdução Genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Down-regulation of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) tumor suppressor gene expression is common in several malignancies including prostate, colon, and breast cancer. The mechanism that mediates this down-regulation is not known. Here, we report that down-regulation of CEACAM1 expression in prostate cancer cells occurs primarily at the transcriptional level and is mediated by Sp2, a member of the Sp family of transcription factors. Sp2 binds to the CEACAM1 promoter in vitro and in vivo, and transient overexpression of Sp2 down-regulates endogenous CEACAM1 expression in normal prostate epithelial cells. Sp2 appears to repress CEACAM1 gene expression by recruiting histone deacetylase activity to the CEACAM1 promoter. In human prostate cancer specimens, Sp2 expression is high in prostate cancer cells but low in normal prostate epithelial cells and is inversely correlated with CEACAM1 expression. Our studies show that transcriptional repression by Sp2 represents one mechanism by which CEACAM1 tumor suppressor gene is down-regulated in prostate cancer.
Assuntos
Antígenos CD/genética , Antígenos de Diferenciação/genética , Proteínas de Ligação a DNA/genética , Neoplasias da Próstata/genética , Fatores de Transcrição/genética , Acetiltransferases/antagonistas & inibidores , Animais , Antígenos CD/biossíntese , Antígenos de Diferenciação/biossíntese , Antígeno Carcinoembrionário , Moléculas de Adesão Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica , Histona Acetiltransferases , Humanos , Ácidos Hidroxâmicos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Regiões Promotoras Genéticas , Neoplasias da Próstata/metabolismo , Fator de Transcrição Sp2 , Fatores de Transcrição/biossíntese , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
OBJECTIVE: A diabetic mouse model of accelerated neointimal formation would be a useful tool to understand the increased incidence of restenosis in patients with diabetes. METHODS AND RESULTS: Femoral artery endoluminal wire injury was performed in diabetic insulin 2 Akita (ins2Akita) and leptin receptor db/db (leprdb/db) mutant mice. Neointima size in ins2Akita mouse arteries was unchanged compared with nondiabetic wild-type littermates. Although Ki67 labeling demonstrated similar rates of replication in the neointima of leprdb/db mouse arteries, neointimal formation in leprdb/db mice was surprisingly reduced by approximately 90% compared with nondiabetic lepr+/+ mice. Four hours after arterial injury, medial smooth muscle cell death was diminished in leprdb/db arteries, suggesting that the initial response to arterial injury was altered in leprdb/db mice. CONCLUSIONS: These studies highlight a differential response to arterial injury in leprdb/db mice and suggest a potential role for leptin in the regulation of neointimal formation in response to arterial injury.
Assuntos
Diabetes Mellitus Tipo 1/fisiopatologia , Diabetes Mellitus Tipo 2/fisiopatologia , Leptina/fisiologia , Músculo Liso Vascular/lesões , Neovascularização Patológica/fisiopatologia , Túnica Íntima/patologia , Túnica Íntima/fisiopatologia , Animais , Reestenose Coronária/fisiopatologia , Modelos Animais de Doenças , Feminino , Artéria Femoral/lesões , Artéria Femoral/patologia , Artéria Femoral/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Fatores Sexuais , Túnica Média/patologiaRESUMO
Adult stem cells have potential use for several biomedical applications, including cell replacement therapy, gene therapy, and tissue engineering. However, such applications have been limited due to difficulties encountered in expanding functional adult stem cells. We have developed a new approach to the problem of adult stem cell expansion based on the suppression of asymmetric cell kinetics (SACK). We postulated that asymmetric cell kinetics, required for adult stem cell function, were a major barrier to their expansion in culture. As such, conversion of adult stem cells from asymmetric cell kinetics to symmetric cell kinetics would promote their exponential expansion and longterm propagation in culture. The purine nucleoside xanthosine (Xs), which promotes guanine ribonucleotide biosynthesis, can be used to reversibly convert cells from asymmetric cell kinetics to symmetric cell kinetics. We used Xs supplementation to derive clonal epithelial cell lines from adult rat liver that have properties of adult hepatic stem cells. The properties of two Xs-derived cell lines, Lig-8 and Lig-13, are described in detail and compared to properties of adult rat hepatic cell lines derived without Xs supplementation. The Xs-derived cell lines exhibit Xs-dependent asymmetric cell kinetics and Xs-dependent expression of mature hepatic differentiation markers. Interestingly, Lig-8 cells produce progeny with properties consistent with hepatocyte differentiation, while Lig-13 progeny cells have properties consistent with bile duct epithelium differentiation. A stable adult cholangiocyte stem cell line has not been previously described. Consistent with the principles of their derivation, the SACK-derived hepatic cell lines exhibit neither senescence nor tumorigenic properties, and their differentiation properties are stable after longterm culture. These characteristics of SACK-derived stem cell lines underscore asymmetric cell kinetics as an essential adult stem cell property with potential to be the basis for a general approach to expansion and propagation of diverse adult stem cells.
Assuntos
Células Clonais , Células Epiteliais/fisiologia , Ribonucleosídeos/metabolismo , Células-Tronco/citologia , Células-Tronco/fisiologia , Animais , Ductos Biliares/citologia , Biomarcadores , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Células Cultivadas , Hepatócitos/citologia , Hepatócitos/fisiologia , Cinética , Masculino , Modelos Biológicos , Ratos , Ratos Endogâmicos F344 , XantinasRESUMO
The 16-kDa prolactin (PRL), derived from the proteolytic cleavage of wild-type 23-kDa PRL, has been shown to have antiangiogenic activity. Such an antiangiogenic activity may have an effect on tumor growth in vivo. Here we examined the effect of 23-kDa and 16-kDa PRL on tumor growth, and the potential of using recombinant 16-kDa human PRL for prostate cancer therapy. The effects of 23-kDa PRL and 16-kDa PRL on the tumorigenicity of prostate cancer cells in vivo were studied. Using an adenovirus transfer vector to achieve high efficiency 23-kDa and 16-kDa PRL transfection in DU145 and PC-3 human prostate carcinoma cell lines, we demonstrated that expression of 16-kDa PRL in the prostate cancer cells markedly reduced their ability to form tumors in a xenograft animal model. These studies established that the 16-kDa PRL has antitumor activity in vivo, presumably as a result of its antiangiogenic effect. Interestingly, 23-kDa PRL showed a weak and transient suppression of prostate tumor growth. The weak antitumor activity of 23-kDa PRL may be because of the production of 16-kDa PRL from 23-kDa PRL by the tumor cells. Thus, the apparent effect of 23-kDa PRL on the growth of DU145 and PC-3 cells in vivo may result from the combined effects of 23-kDa PRL and 16-kDa PRL. These results suggest that the 16-kDa PRL has potential as a treatment agent in prostate cancer.
Assuntos
Terapia Genética/métodos , Fragmentos de Peptídeos/genética , Prolactina/genética , Neoplasias da Próstata/terapia , Adenoviridae/genética , Animais , Divisão Celular/genética , Endotélio Vascular/patologia , Expressão Gênica , Vetores Genéticos/genética , Humanos , Masculino , Camundongos , Camundongos Nus , Fragmentos de Peptídeos/biossíntese , Prolactina/biossíntese , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Transfecção , Transgenes , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A long-standing intriguing hypothesis in cancer biology is that adult stem cells avoid mutations from DNA replication errors by a unique pattern of chromosome segregation. At each asymmetric cell division, adult stem cells have been postulated to selectively retain a set of chromosomes that contain old template DNA strands (i.e., "immortal DNA strands"). Using cultured cells that cycle with asymmetric cell kinetics, we confirmed both the existence of immortal DNA strands and the cosegregation of chromosomes that bear them. Our findings also lead us to propose a role for immortal DNA strands in tissue aging as well as cancer.