RESUMO
Radiolabeled peptides with high specificity for overexpressed receptors in tumor cells hold great promise for diagnostic and therapeutic applications. In this work, we aimed at comparing the radiolabeling efficiency and biological properties of two different RGD analogs: GRGDYV and GRGDHV, labeled with iodine-131 (131I) and technetium-99m-tricarbonyl complex [99mTc][Tc(CO)3]+. Additionally, we evaluated their interaction with the αvß3 integrin molecule, overexpressed in a wide variety of tumors, including glioblastoma. Both peptides were chemically synthesized, purified and radiolabeled with 131I and [99mTc][Tc(CO)3]+ using the chloramine-T and tricarbonyl methodologies, respectively. The stability, binding to serum proteins and partition coefficient were evaluated for both radioconjugates. In addition, the binding and internalization of radiopeptides to rat C6 glioblastoma cells and rat brain homogenates from normal animals and a glioblastoma-induced model were assessed. Finally, ex vivo biodistribution studies were carried out. Radiochemical yields between 95-98% were reached for both peptides under optimized radiolabeling conditions. Both peptides were stable for up to 24 h in saline solution and in human serum. In addition, the radiopeptides have hydrophilic characteristics and a percentage of binding to serum proteins around 35% and 50% for the [131I]I-GRGDYV and [99mTc]Tc(CO)3-GRGDHV fragments, respectively. Radiopeptides showed the capacity of binding and internalization both in cell culture (C6) and rat brain homogenates. Biodistribution studies corroborated the results obtained with brain homogenates and confirmed the different binding characteristics due to the exchange of radionuclides and the presence of the tricarbonyl complex. Thereby, the results showed that both radiopeptides might be considered for future clinical applications.
RESUMO
Radiolabeled peptides with high specificity to receptors expressed on tumor cells hold a great promise as diagnostic and therapeutic tracers. The main objective of this study was to evaluate the radiochemical and biological properties of two [131I]I-peptides, as well as their interaction with the epidermal growth factor receptor (EGFR), overexpressed in a wide variety of tumors, including glioblastoma. The EEEEYFELV peptide and its analogue DEDEYFELV, both designed to interact with EGFR, were chemically synthesized, purified and radiolabeled with iodine-131 ([131I]NaI). The radioiodination was evaluated and optimized using the chloramine-T methodology. The stability, serum proteins binding and partition coefficient were assessed for both radioconjugates. Moreover, the binding and internalization of synthesized radiopeptides with rat glioblastoma cells (C6) and with rat brain homogenates from a glioblastoma induced model were evaluated and ex vivo biodistribution studies were performed. Under optimized radiolabeling conditions, the peptides showed an average radiochemical yield of 90-95%. The stability studies showed that both peptides were stable up to 24 h in reaction medium, saline, and human serum. Furthermore, [131I]I-peptides have hydrophilic features and showed binding percentage to serum proteins of around 50%, which is highly compatible with clinical applications. Moreover, the radiopeptides presented capacity for binding and internalization in both tumor cells (C6) and rat brain tissues after tumor induction. Biodistribution studies corroborated the cell culture studies and confirmed the different binding characteristics derived from a simple change of two amino acids (Glu â Asp1,3) in their sequences. The results obtained are consistent enough to motivate further studies. Thereby, these radiolabeled peptides might be useful for diagnostic applications.
Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Radioisótopos do Iodo/farmacocinética , Fragmentos de Peptídeos/metabolismo , Compostos Radiofarmacêuticos/farmacocinética , Animais , Apoptose , Neoplasias Encefálicas/metabolismo , Proliferação de Células , Receptores ErbB/metabolismo , Glioblastoma/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Searching for targets that allow pharmacological inhibition of cell proliferation in over-proliferative states, such as cancer, leads us to finely understand the complex mechanisms orchestrating the perfect control of mitosis number, frequency and pace as well as the molecular arrangements that induce cells to enter functional quiescence and brings them back to cycling in specific conditions. Although the mechanisms regulating cell proliferation have been described several years ago, never before has so much light been shed over this machinery as during the last decade when therapy targets have been explored and molecules, either synthetic or in the form of antibodies with the potential of becoming cancer drugs were produced and adjusted for specific binding and function. Proteins containing tyrosine kinase domains, either membrane receptors or cytoplasmic molecules, plus the ones activated by those in downstream pathways, having tyrosine kinase domains or not, such as RAS which is a GTPase and serine/threonine kinases such as RAF, play crucial role in conducting proliferation information from cell surroundings to the nucleus where gene expression takes place. Tyrosine kinases phosphorylate tyrosine residues in an activating mode and are found in important growth factor receptors, such as for ligands from families collectively known as VEGF, PDGF and EGF, to name a few and in intracellular downstream molecules. They all play important roles in normal physiology and are commonly found mutated or overexpressed in neoplastic states. Our objective here is to present such kinases as druggable targets for cancer therapy, highlighting the ones for which the pharmacological arsenal is available, discussing specificity, resistance mechanisms and treatment alternatives in cases of resistance, plus listing potential targets that have not been successfully worked yet.
Assuntos
Neoplasias/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais , Ensaios Clínicos como Assunto , Descoberta de Drogas , Resistencia a Medicamentos Antineoplásicos , Humanos , Terapia de Alvo Molecular , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Ligação Proteica , Inibidores de Proteínas Quinases/uso terapêutico , Receptores Proteína Tirosina Quinases/genéticaRESUMO
Cancers derive from step by step processes which are differentiated by the progressively accumulated mutations. For some tumors there is a clear progressive advancement from benign lesions to malignancy and for these, preventive screening programs exist. In such cases having those benign lesions are a clear indicator of predisposition while for some other cases, familial patterns of cancer incidence and the identification of mutations are the main indicators of higher risk for having the disease. For patients identified as having predisposition, chemoprevention is a goal and in some cases a possibility. Chemoprevention is the use of any compound, either natural or synthetic that abrogates carcinogenesis or tumor progression, through different mechanisms, some of which have already been described. For example, the classic mechanisms may involve activation of free radical scavenging enzymes, control of chronic inflammation, and downregulation of specific signaling pathways. More recently, epigenetics allowed further understanding of the chemopreventive potential of several agents, such as sulforaphane, green tea derived compounds, resveratrol, isoflavones, and others which we exploit in this review article. Throughout the text we discuss the properties compounds should have in order to be classified as chemopreventive ones and the challenges in translational research in this area, as lots of the success achieved in vitro cannot be translated into the clinical settings, due to several different drawbacks, which include toxicity, cost, dose definition, patient adherence, and regimen of use.
RESUMO
The purpose of this investigation was to determine lipid peroxidation markers, physiological stress and muscle damage in elite kayakers in response to a maximum 4-min kayak ergometer test (KE test), and possible correlations with individual 1000m kayaking performances. The sample consisted of twenty-three adult male and nine adult female elite kayakers, with more than three years' experience in international events, who voluntarily took part in this study. The subjects performed a 10-min warm-up, followed by a 2-min passive interval, before starting the test itself, which consisted of a maximum 4-min work paddling on an ergometer; right after the end of the test, an 8 ml blood sample was collected for analysis. 72 hours after the test, all athletes took part in an official race, when then it was possible to check their performance in the on site K1 1000m test (P1000m). The results showed that all lipoproteins and hematological parameters tested presented a significant difference (p≤0.05) after exercise for both genders. In addition, parameters related to muscle damage such as lactate dehydrogenase (LDH) and creatine kinase (CK) presented significant differences after stress. Uric acid presented an inverse correlation with the performance (r = -0.76), while CK presented a positive correlation (r = 0.46) with it. Based on these results, it was possible to verify muscle damage and the level of oxidative stress caused by indoor training with specific ergometers for speed kayaking, highlighting the importance of analyzing and getting to know the physiological responses to this type of training, in order to provide information to coaches and optimize athletic performance.
Assuntos
Teste de Esforço , Músculo Esquelético/fisiopatologia , Estresse Oxidativo , Esportes , Adulto , Feminino , Humanos , MasculinoRESUMO
The objective of this study was to evaluate the correlation between cyclooxygenase-2 (COX-2) and markers of cell proliferation and apoptosis, including, Bcl-2, Bax, Ki-67 and the type I insulin-like growth factor (IGF) receptor (IGF1-R) in ductal carcinoma in situ (DCIS) and infiltrating ductal carcinoma (IDC), present in the same surgical specimen. A total of 110 cases were evaluated using tissue microarrays. Cases were classified in scores from 0 to 3 according to pre-defined methods. The results showed that the positivity rates were COX-2 in 87% of cases in DCIS and IDC; Bcl-2 in 55% of cases in DCIS and IDC; Bax in 23% of cases in IDC and 19% in DCIS, IGF-1 in 24% of cases in DCIS and IDC; and Ki-67 in 81% of cases in DCIS and IDC. We also observed a positive correlation between the expression of COX-2 and IGF1-R (p=0.045). Our results demonstrate a positive correlation between the expression of COX-2 and IGF1-R in DCIS and IDC, demonstrating that they are involved in breast cancer carcinogenesis. Further studies are required to prove the effectiveness of COX-2 and IGF1-R inhibitors for the prevention and treatment of breast cancer, as well as to explain their mechanism of action.
RESUMO
CONTEXT AND OBJECTIVE: Cyclooxygenase-2 (COX-2) and human epidermal growth factor receptor type 2 (HER-2) are associated with tumorigenesis. Studies have shown that HER-2 can regulate COX-2 expression. The aim of this study was to evaluate the correlation between COX-2 and HER-2 expression in normal breast epithelium and in ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) present in the same breast. DESIGN AND SETTING: Cross-sectional study at the Mastology Unit of the Department of Gynecology and Obstetrics, Santa Casa de Misericórdia de São Paulo Hospital. METHODS: COX-2 and HER-2 were detected using immunohistochemistry on 100 tissue fragments. HER-2 > +2 was subjected to fluorescence in situ hybridization (FISH). RESULTS: COX-2 expression was detected in 87 percent, 85 percent and 75 percent of IDC, DCIS and normal epithelium, respectively. HER-2 expression was detected in 34 percent of IDC and 34 percent of DCIS. COX-2 in DCIS correlated with HER-2 in IDC (P = 0.049) and DCIS (P = 0.049). COX-2 in normal epithelium correlated with HER-2 in IDC (P = 0.046) and DCIS (P = 0.046). COX-2 in IDC was not associated with HER-2 (P = 0.235). Comparison between COX-2 and HER-2 in DCIS showed that there was a statistically significant difference with regard to nuclear grades II and III and presence of comedonecrosis (P < 0.001). In IDC, there was significant expression with nuclear grades II and III and histological grade II (P < 0.001). CONCLUSIONS: Our findings provide evidence that HER-2 and COX-2 regulate each other.
CONTEXTO E OBJETIVO: Ciclo-oxigenase (COX-2) e receptor tipo 2 do fator de crescimento epidérmico humano (HER-2) estão associados com tumorigênese. Estudos mostraram que HER-2 pode regular a expressão de COX-2. O objetivo deste estudo foi avaliar a correlação entre expressão da COX-2 e HER-2 no epitélio normal de mama, no carcinoma ductal in situ (DCIS) e carcinoma ductal invasivo (IDC) presentes na mesma mama. TIPO DE ESTUDO E LOCAL: Estudo transversal na clínica de Mastologia do Departamento de Obstetrícia e Ginecologia do Hospital da Santa Casa de Misericórdia de São Paulo. MÉTODOS: A detecção da COX-2 e HER-2 foi realizada por imunoistoquímica em 100 fragmentos teciduais. HER-2 > +2 foi submetido a hibridização fluorescente in situ (FISH). RESULTADOS: Expressão de COX-2 foi detectada em 87 por cento, 85 por cento e 75 por cento dos IDC, DCIS e epitélio normal, respectivamente. Expressão de HER-2 foi detectada em 34 por cento dos IDC e 34 por cento de DCIS. COX-2 em DCIS correlacionou-se com HER-2 em IDC (P = 0,049) e DCIS (P = 0,049). COX-2 no epitélio normal correlacionou-se com HER-2 em IDC (P = 0,046) e DCIS (P = 0,046). COX-2 no IDC não foi associada com HER-2 (P = 0,235). Quando comparado COX-2 com HER-2 em DCIS houve diferença estatisticamente significante com relação ao grau nuclear II e III e presença de comedonecrose (P < 0,001) e no IDC, houve expressão significativa no grau nuclear II e III e histológico II (P < 0,001). CONCLUSÕES: Nossos achados mostram evidências que HER-2 e COX-2 se autorregulam.
Assuntos
Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Pessoa de Meia-Idade , Neoplasias da Mama/enzimologia , Carcinoma Ductal de Mama/enzimologia , Carcinoma Intraductal não Infiltrante/enzimologia , /metabolismo , Proteínas de Neoplasias/metabolismo , /metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Carcinoma Intraductal não Infiltrante/patologia , Imuno-Histoquímica , Necrose , Regulação para CimaRESUMO
CONTEXT AND OBJECTIVE: Cyclooxygenase-2 (COX-2) and human epidermal growth factor receptor type 2 (HER-2) are associated with tumorigenesis. Studies have shown that HER-2 can regulate COX-2 expression. The aim of this study was to evaluate the correlation between COX-2 and HER-2 expression in normal breast epithelium and in ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) present in the same breast. DESIGN AND SETTING: Cross-sectional study at the Mastology Unit of the Department of Gynecology and Obstetrics, Santa Casa de Misericórdia de São Paulo Hospital. METHODS: COX-2 and HER-2 were detected using immunohistochemistry on 100 tissue fragments. HER-2 > +2 was subjected to fluorescence in situ hybridization (FISH). RESULTS: COX-2 expression was detected in 87%, 85% and 75% of IDC, DCIS and normal epithelium, respectively. HER-2 expression was detected in 34% of IDC and 34% of DCIS. COX-2 in DCIS correlated with HER-2 in IDC (P = 0.049) and DCIS (P = 0.049). COX-2 in normal epithelium correlated with HER-2 in IDC (P = 0.046) and DCIS (P = 0.046). COX-2 in IDC was not associated with HER-2 (P = 0.235). Comparison between COX-2 and HER-2 in DCIS showed that there was a statistically significant difference with regard to nuclear grades II and III and presence of comedonecrosis (P < 0.001). In IDC, there was significant expression with nuclear grades II and III and histological grade II (P < 0.001). CONCLUSIONS: Our findings provide evidence that HER-2 and COX-2 regulate each other.
Assuntos
Neoplasias da Mama/enzimologia , Carcinoma Ductal de Mama/enzimologia , Carcinoma Intraductal não Infiltrante/enzimologia , Ciclo-Oxigenase 2/metabolismo , Proteínas de Neoplasias/metabolismo , Receptor ErbB-2/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Carcinoma Intraductal não Infiltrante/patologia , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Necrose , Regulação para CimaRESUMO
The problem of pancreas donor shortage could be addressed through in vitro islet-cell proliferation prior to transplantation into diabetic patients. Therefore, we set out to evaluate the effects of prolactin (rhPRL) and laminin on primary cultures of human pancreatic islets. Our results showed that rhPRL induced an increase in islet-cell number and in cumulative insulin secretion (p<0.01). However, glucose-induced insulin secretion was enhanced only in the presence of both laminin and rhPRL. In addition, we describe, for the first time in human islets, the PRL-induced activation of JAK2, and signal transducer and activator of transcription (STAT) 1, 3 and 5. Our results demonstrate a significant beneficial effect of rhPRL and laminin on human islets and support widely held notion that the closer physiological stimuli and environment of beta cells are mimicked, the better are the results in cell proliferation and secretory function, both essential for successful islet transplantation.
Assuntos
Ilhotas Pancreáticas/efeitos dos fármacos , Laminina/farmacologia , Prolactina/farmacologia , Adulto , Idoso , Western Blotting , Proliferação de Células/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Imunofluorescência , Glucose/farmacologia , Humanos , Imunoprecipitação , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Janus Quinase 2/metabolismo , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição STAT/metabolismoRESUMO
Glucocorticoid hormones (GCs) exert a potent anti-proliferative activity on several cell types. The classic molecular mechanism of GCs involves modulation of the activity of the glucocorticoids receptor, a transcriptional regulator. However, the anti-proliferative effect of GCs may also involve modulation of processes such as translation, subcellular localization and post-translational modifications, which are not reflected at the mRNA level. To investigate these potential effects of GCs, we employed the proteomic approach (two-dimensional electrophoresis and mass spectrometry) and the ST1 cells, obtained from the C6 rat glioma cell line, as a model. GC treatment leads ST1 cells to a complete transformed-to-normal phenotypic reversion and loss of their tumorigenic potential. By comparing sets of 2D nuclear protein profiles of ST1 cells treated (or not) with hydrocortisone (Hy), 13 polypeptides displaying >or=two-fold difference in abundance upon Hy treatment were found. Five of these polypeptides were identified by peptide mass fingerprinting, including Annexin 2 (ANX2), hnRNP A3 and Ubiquitin. Evidence obtained by Western blot analysis indicates that ANX2 is present in the nucleus and has its subcellular localization modulated by GC-treatment of ST1 cells. Our findings indicate complementary mechanisms contributing to the regulation of gene expression associated with ST1 cells' response to GCs.
Assuntos
Proliferação de Células/efeitos dos fármacos , Glioma/patologia , Glucocorticoides/farmacologia , Proteômica , Animais , Anexina A2/análise , Anexina A2/metabolismo , Hidrocortisona/farmacologia , Espectrometria de Massas , Proteínas Nucleares/análise , Mapeamento de Peptídeos/métodos , Ratos , Células Tumorais CultivadasRESUMO
Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinemia. Leptin has been implicated as an antiapoptotic compound as well as a stimulant of the immune response. Leptin administration is capable of reversing the immune deficiency that occurs upon starvation. We investigated a possible role for leptin in CVID; a condition associated with lowered plasma leptin levels. Thirty-eight patients were studied. Addition of leptin to the tissue culture media of PBMC from CVID patients increased the proliferative response of lymphocytes to mitogens and decreased activation-induced apoptosis of these cells. IL-2 and specially IL-4 production also increased significantly upon addition of leptin to the PBMC cultures. Our results suggest that leptin may be involved in some of the cellular defects observed in CVID and indicate a novel therapeutic strategy to improve immune function in these patients.
Assuntos
Imunodeficiência de Variável Comum/tratamento farmacológico , Imunodeficiência de Variável Comum/imunologia , Interleucina-2/biossíntese , Interleucina-4/biossíntese , Leptina/uso terapêutico , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Adolescente , Adulto , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Estudos de Coortes , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Interleucina-2/imunologia , Interleucina-4/imunologia , Modelos Lineares , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Linfócitos T/citologiaRESUMO
Controlar a proliferação celular de tumores é um objetivo que vem sendo perseguido há décadas, com moderado sucesso na maioria dos casos. Dentre os diversos tipos de tumores que atingem a humanidade, alguns gliomas são considerados os mais fatais, por haver pouca ou nenhuma alternativa de tratamento efetivo. Agentes que apresentam propriedades anti-tumorais, como glicocorticóides (GC) e a forma all-trans do ácido retinóico (ATRA) são utilizados como adjuvantes no tratamento de alguns tipos de glioma. Entretanto, apesar de serem moléculas bastante conhecidas, pouco se sabe sobre seu mecanismo de ação como anti-tumoral. Para endereçar este problema, nosso laboratório se propôs a isolar e caracterizar genes regulados por estes agentes, utilizando modelos celulares, como as linhagens C6 e ST1 de glioma de rato, e as linhagens T98G e A172 de glioma humano...