RESUMO
In general, detection of peritoneal carcinomatosis (PC) occurs at the late stage when there is no treatment option. In the present study, we designed novel drug delivery systems that are functionalized with anti-CD133 antibodies. The C1, C2 and C3 complexes with cisplatin were introduced into nanotubes, either physically or chemically. The complexes were reacted with anti-CD133 antibody to form the labeled product of A0-o-CX-chem-CD133. Cytotoxicity screening of all the complexes was performed on CHO cells. Data showed that both C2 and C3 Pt-complexes are more cytotoxic than C1. Flow-cytometry analysis showed that nanotubes conjugated to CD133 antibody have the ability to target cells expressing the CD133 antigen which is responsible for the emergence of resistance to chemotherapy and disease recurrence. The shortest survival rate was observed in the control mice group (K3) where no hyperthermic intraperitoneal chemotherapy procedures were used. On the other hand, the longest median survival rate was observed in the group treated with A0-o-C1-chem-CD133. In summary, we designed a novel drug delivery system based on carbon nanotubes loaded with Pt-prodrugs and functionalized with anti-CD133 antibodies. Our data demonstrates the effectiveness of the new drug delivery system and provides a novel therapeutic modality in the treatment of melanoma.
Assuntos
Cisplatino/administração & dosagem , Cisplatino/química , Sistemas de Liberação de Medicamentos/métodos , Hipertermia Induzida/métodos , Nanotubos de Carbono/química , Neoplasias Peritoneais/terapia , Antígeno AC133 , Animais , Anticorpos/administração & dosagem , Anticorpos/química , Anticorpos/imunologia , Antígenos CD/química , Antígenos CD/imunologia , Terapia Combinada , Modelos Animais de Doenças , Glicoproteínas/química , Glicoproteínas/imunologia , Imunotoxinas/administração & dosagem , Imunotoxinas/química , Imunotoxinas/imunologia , Injeções Intraperitoneais , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/química , Peptídeos/imunologia , Neoplasias Peritoneais/tratamento farmacológico , Taxa de SobrevidaRESUMO
The balance of nitric oxide (NO) versus superoxide generation has a major role in the initiation and progression of endothelial dysfunction. Under conditions of high glucose, endothelial nitric oxide synthase (eNOS) functions as a chief source of superoxide rather than NO. In order to improve NO bioavailability within the vessel wall in type-1 diabetes, we investigated treatment strategies that improve eNOS phosphorylation and NO-dependent vasorelaxation. We evaluated methods to increase the eNOS activity by (1) feeding Ins2(Akita) spontaneously diabetic (type-1) mice with l-arginine in the presence of sepiapterin, a precursor of tetrahydrobiopterin; (2) preventing eNOS/NO deregulation by the inclusion of inhibitor kappa B kinase beta (IKKß) inhibitor, salsalate, in the diet regimen in combination with l-arginine and sepiapterin; and (3) independently increasing eNOS expression to improve eNOS activity and associated NO production through generating Ins2(Akita) diabetic mice that overexpress human eNOS predominantly in vascular endothelial cells. Our results clearly demonstrated that diet supplementation with l-arginine, sepiapterin along with salsalate improved phosphorylation of eNOS and enhanced vasorelaxation of thoracic/abdominal aorta in type-1 diabetic mice. More interestingly, despite the overexpression of eNOS, the in-house generated transgenic eNOS-GFP (TgeNOS-GFP)-Ins2(Akita) cross mice showed an unanticipated effect of reduced eNOS phosphorylation and enhanced superoxide production. Our results demonstrate that enhancement of endogenous eNOS activity by nutritional modulation is more beneficial than increasing the endogenous expression of eNOS by gene therapy modalities.
Assuntos
Diabetes Mellitus Tipo 1/prevenção & controle , Suplementos Nutricionais , Endotélio Vascular/metabolismo , Hipoglicemiantes/uso terapêutico , Óxido Nítrico Sintase Tipo III/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Processamento de Proteína Pós-Traducional , Animais , Aorta/citologia , Aorta/metabolismo , Aorta/fisiopatologia , Arginina/metabolismo , Arginina/uso terapêutico , Bovinos , Células Cultivadas , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/fisiopatologia , Endotélio Vascular/citologia , Endotélio Vascular/fisiopatologia , Feminino , Heterozigoto , Humanos , Hipoglicemiantes/metabolismo , Insulina/genética , Insulina/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Óxido Nítrico Sintase Tipo III/química , Óxido Nítrico Sintase Tipo III/genética , Fosforilação , Inibidores de Proteínas Quinases/metabolismo , Pterinas/metabolismo , Pterinas/uso terapêutico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Salicilatos/metabolismo , Salicilatos/uso terapêutico , DesmameRESUMO
BACKGROUND: Deficiency in tuberin results in activation the mTOR pathway and leads to accumulation of cell matrix proteins. The mechanisms by which tuberin regulates fibrosis in kidney angiomyolipomas (AMLs) of tuberous sclerosis patients are not fully known. METHOD: In the present study, we investigated the potential role of tuberin/mTOR pathway in the regulation of cell fibrosis in AML cells and kidney tumor tissue from tuberous sclerosis complex (TSC) patients. RESULTS: AML cells treated with rapamycin shows a significant decrease in mRNA and protein expression as well as in promoter transcriptional activity of alpha-smooth muscle actin (α-SMA) compared to untreated cells. In addition, cells treated with rapamycin significantly decreased the protein expression of the transcription factor YY1. Rapamycin treatment also results in the redistribution of YY1 from the nucleus to cytoplasm in AML cells. Moreover, cells treated with rapamycin resulted in a significant reduce of binding of YY1 to the αSMA promoter element in nuclear extracts of AML cells. Kidney angiomyolipoma tissues from TSC patients showed lower levels of tuberin and higher levels of phospho-p70S6K that resulted in higher levels of mRNA and protein of αSMA expression compared to control kidney tissues. In addition, most of the α-SMA staining was identified in the smooth muscle cells of AML tissues. YY1 was also significantly increased in tumor tissue of AMLs compared to control kidney tissue suggesting that YY1 plays a major role in the regulation of αSMA. CONCLUSIONS: These data comprise the first report to provide one mechanism whereby rapamycin might inhibit the cell fibrosis in kidney tumor of TSC patients.
Assuntos
Neoplasias Renais/complicações , Neoplasias Renais/patologia , Esclerose Tuberosa/complicações , Actinas/genética , Actinas/metabolismo , Linhagem Celular , Ativação Enzimática , Fibrose , Regulação da Expressão Gênica , Humanos , Espaço Intracelular/metabolismo , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Modelos Biológicos , Mutação , Regiões Promotoras Genéticas , Ligação Proteica , Transporte Proteico , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/metabolismo , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismoRESUMO
Apoptosis contributes to the development of diabetic nephropathy, but the mechanism by which high glucose (HG) induces apoptosis is not fully understood. Because the tuberin/mTOR pathway can modulate apoptosis, we studied the role of this pathway in apoptosis in type I diabetes and in cultured proximal tubular epithelial (PTE) cells exposed to HG. Compared with control rats, diabetic rats had more apoptotic cells in the kidney cortex. Induction of diabetes also increased phosphorylation of tuberin in association with mTOR activation (measured by p70S6K phosphorylation), inactivation of Bcl-2, increased cytosolic cytochrome c expression, activation of caspase 3, and cleavage of PARP; insulin treatment prevented these changes. In vitro, exposure of PTE cells to HG increased phosphorylation of tuberin and p70S6K, phosphorylation of Bcl-2, expression of cytosolic cytochrome c, and caspase 3 activity. High glucose induced translocation of the caspase substrate YY1 from the cytoplasm to the nucleus and enhanced cleavage of PARP. Pretreatment the cells with the mTOR inhibitor rapamycin reduced the number of apoptotic cells induced by HG and the downstream effects of mTOR activation noted above. Furthermore, gene silencing of tuberin with siRNA decreased cleavage of PARP. These data show that the tuberin/mTOR pathway promotes apoptosis of tubular epithelial cells in diabetes, mediated in part by cleavage of PARP by YY1.