RESUMO
OBJECTIVE: Targeting the glucagon-like peptide-1 receptor with radiolabeled exendin is a very promising method to noninvasively determine the ß cell mass in the pancreas, which is needed to unravel the pathophysiology of type 1 and type 2 diabetes. The present study aimed to explore the effects of both hyperglycemia and insulitis on the uptake of exendin in a spontaneous type 1 diabetes mouse model, nonobese diabetic (NOD) mice. METHODS: NOD mice (n = 75, 7-21 weeks old) were injected intravenously with [111In]In-DTPA-exendin-3, and single-photon emission computed tomography (SPECT) images were acquired 1 h pi. The pancreatic accumulation of [111In]In-DTPA-exendin-3 was quantified in vivo using SPECT and by ex vivo counting and correlated to the ß cell mass (BCM). The influence of insulitis and hyperglycemia on the exendin uptake was assessed. RESULTS: The pancreas could be visualized longitudinally using SPECT. A linear correlation was found between the BCM (%) and pancreatic uptake (%ID/g) as measured by ex vivo counting (Pearson r = 0.64, p < 0.001), which was not affected by either insulitis (Pearson r = 0.66, p = 0.83) or hyperglycemia (Pearson r = 0.57, p = 0.51). Biodistribution and ex vivo autoradiography revealed remaining [111In]In-DTPA-exendin-3 uptake in the pancreas despite total ablation of BCM. CONCLUSIONS: Despite hyperglycemia and severe insulitis, we have found a good correlation between BCM and pancreatic exendin uptake, even in a suboptimal model with relatively high background activity.
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Hiperglicemia/metabolismo , Células Secretoras de Insulina/metabolismo , Peptídeos/metabolismo , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Animais , Autorradiografia , Diabetes Mellitus Tipo 1/diagnóstico por imagem , Modelos Animais de Doenças , Feminino , Imuno-Histoquímica , Radioisótopos de Índio/administração & dosagem , Radioisótopos de Índio/química , Radioisótopos de Índio/metabolismo , Injeções Intravenosas , Camundongos , Camundongos Endogâmicos NOD , Ácido Pentético/administração & dosagem , Ácido Pentético/química , Ácido Pentético/metabolismo , Peptídeos/administração & dosagem , Peptídeos/química , Compostos Radiofarmacêuticos/metabolismo , Distribuição TecidualRESUMO
The changes in ß-cell mass (BCM) during the development and progression of diabetes could potentially be measured by radionuclide imaging using radiolabeled exendin. In this study, we investigated the potential of 111In-diethylenetriaminepentaacetic acid-exendin-3 (111In-exendin) in a rat model that closely mimics the development of type 1 diabetes (T1D) in humans: BioBreeding diabetes-prone (BBDP) rats. BBDP rats of 4-18 weeks of age were injected intravenously with 111In-exendin, and single-photon emission computed tomography (SPECT) images were acquired. The accumulation of the radiotracer was measured as well as the BCM and grade of insulitis by histology. 111In-exendin accumulated specifically in the islets, resulting in a linear correlation with the BCM (%) (Pearson r = 0.89, P < 0.0001, and r = 0.64 for SPECT). Insulitis did not have an influence on this correlation. These results indicate that 111In-exendin is a promising tracer to determine the BCM during the development of T1D, irrespective of the degree of insulitis.
Assuntos
Radioisótopos de Índio/análise , Células Secretoras de Insulina/metabolismo , Peptídeos/análise , Animais , Diabetes Mellitus Tipo 1/metabolismo , Feminino , Humanos , Ratos , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
Expression of EGFRvIII is frequently observed in glioblastoma and is associated with increased cellular proliferation, enhanced tolerance to metabolic stresses, accelerated tumor growth, therapy resistance and poor prognosis. We observed that expression of EGFRvIII elevates the activation of macroautophagy/autophagy during starvation and hypoxia and explored the underlying mechanism and consequence. Autophagy was inhibited (genetically or pharmacologically) and its consequence for tolerance to metabolic stress and its therapeutic potential in (EGFRvIII+) glioblastoma was assessed in cellular systems, (patient derived) tumor xenopgrafts and glioblastoma patients. Autophagy inhibition abrogated the enhanced proliferation and survival advantage of EGFRvIII+ cells during stress conditions, decreased tumor hypoxia and delayed tumor growth in EGFRvIII+ tumors. These effects can be attributed to the supporting role of autophagy in meeting the high metabolic demand of EGFRvIII+ cells. As hypoxic tumor cells greatly contribute to therapy resistance, autophagy inhibition revokes the radioresistant phenotype of EGFRvIII+ tumors in (patient derived) xenograft tumors. In line with these findings, retrospective analysis of glioblastoma patients indicated that chloroquine treatment improves survival of all glioblastoma patients, but patients with EGFRvIII+ glioblastoma benefited most. Our findings disclose the unique autophagy dependency of EGFRvIII+ glioblastoma as a therapeutic opportunity. Chloroquine treatment may therefore be considered as an additional treatment strategy for glioblastoma patients and can reverse the worse prognosis of patients with EGFRvIII+ glioblastoma.
Assuntos
Autofagia/fisiologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Receptores ErbB/biossíntese , Glioblastoma/metabolismo , Glioblastoma/patologia , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Proliferação de Células , Cloroquina/farmacologia , Cloroquina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/genética , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Humanos , Masculino , Camundongos , Camundongos Nus , Transdução de Sinais , Estresse Fisiológico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Glucagon-like peptide-1 receptor (GLP-1R) targeting using radiolabeled exendin is a promising approach to noninvasively visualize and determine beta cell mass (BCM), which could help to unravel the pathophysiology of diabetes. However, saturation of the GLP-1R on beta cells occurs at low peptide doses, since the number of receptors expressed under physiological conditions is low. Therefore, tracers with high specific activities are required to sensitively image small variations in BCM. Here, we describe a novel exendin-3-based radiotracer with multiple chelators and determine its potential for in vivo beta cell imaging. Exendin-3 was modified by adding six lysine residues C-terminally conjugated with one, two, or six DTPA moieties. All compounds were labeled with 111In and their GLP-1R affinity was determined in vitro using GLP-1R expressing cells. The in vivo behavior of the 111In-labeled tracers was examined in BALB/c nude mice with a subcutaneous GLP-1R expressing tumor (INS-1). Brown Norway rats were used for SPECT visualization of the pancreatic BCM. Addition of six lysine and six DTPA residues (hexendin(40-45)) resulted in a 7-fold increase in specific activity (from 0.73 GBq/nmol to 5.54 GBq/nmol). IC50 values varied between 5.2 and 69.5 nM. All compounds with two or six lysine and DTPA residues had a significantly lower receptor affinity than [Lys40(DTPA)]exendin-3 (4.4 nM, p < 0.05). The biodistribution in mice revealed no significant decrease in pancreatic uptake after addition of six lysine and DTPA molecules. Hexendin(40-45) showed a 6-fold increase in absolute 111In uptake in the pancreas of Brown Norway rats compared to [Lys40(DTPA)]exendin-3 (182.7 ± 42.3 kBq vs 28.8 ± 6.0 kBq, p < 0.001). Visualization of the pancreas on SPECT was improved using hexendin(40-45), due to the higher count rate, achieved at the same peptide dose. In conclusion, hexendin(40-45) showed an improved visualization of the pancreas with SPECT. This tracer holds promise to sensitively and specifically detect small variations in BCM.
Assuntos
Diabetes Mellitus/diagnóstico por imagem , Microscopia Intravital/métodos , Peptídeos/química , Compostos Radiofarmacêuticos/química , Animais , Linhagem Celular Tumoral , Quelantes/química , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Células Secretoras de Insulina , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pâncreas/diagnóstico por imagem , Pâncreas/metabolismo , Ácido Pentético/química , Peptídeos/administração & dosagem , Peptídeos/farmacocinética , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/administração & dosagem , Compostos Radiofarmacêuticos/farmacocinética , Ratos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton Único/métodosRESUMO
BACKGROUND AND PURPOSE: The epidermal growth factor receptor (EGFR) is overexpressed, amplified or mutated in various human epithelial tumors and hypoxia is a common feature of solid tumors. Both EGFR and hypoxia are associated with therapy resistance and poor treatment outcome. To survive hypoxia, cells adapt by activation of hypoxia responsive pathways and expression of hypoxia-induced plasma membrane proteins. We observed that GABAA receptor associated protein like1 (GABARAPL1) and plasma membrane expression of EGFR were increased during hypoxia. Here we explored the role of the GABARAPL1 in EGFR membrane expression during hypoxia. MATERIAL AND METHODS: Quantitative qPCR, immunoblot analysis, flow cytometry and cytochemistry were used to assess this interplay. RESULTS: GABARAPL1 mRNA and protein levels are increased during hypoxia in vitro and correlate with tumor hypoxia in a panel of primary HNSCC xenografts. High GABARAPL1 mRNA is associated with poor outcome of HNSCC patients. During hypoxia, EGFR membrane expression is increased and GABARAPL1 and EGFR colocalize at the plasma membrane. GABARAPL1 knockdown inhibits EGFR membrane expression during hypoxia. CONCLUSION: GABARAPL1 is required for increased membrane expression of EGFR during hypoxia, suggesting a role for GABARAPL1 in the trafficking of these membrane proteins.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Carcinoma de Células Escamosas/metabolismo , Receptores ErbB/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Hipóxia/fisiopatologia , Proteínas Associadas aos Microtúbulos/fisiologia , Antimetabólitos/farmacologia , Hipóxia Celular/fisiologia , Membrana Celular/metabolismo , Movimento Celular/fisiologia , Doxiciclina/farmacologia , Técnicas de Silenciamento de Genes , Humanos , RNA Mensageiro/metabolismo , Células Tumorais CultivadasRESUMO
BACKGROUND: (Pre)clinical studies indicate that autophagy inhibition increases response to anti-cancer therapies. Although promising, due to contradicting reports, it remains unclear if radiation therapy changes autophagy activity and if autophagy inhibition changes the cellular intrinsic radiosensitivity. Discrepancies may result from different assays and models through off-target effects and influencing other signaling routes. In this study, we directly compared the effects of genetic and pharmacological inhibition of autophagy after irradiation in human cancer cell lines. MATERIALS AND METHODS: Changes in autophagy activity after ionizing radiation (IR) were assessed by flux analysis in eight cell lines. Clonogenic survival, DNA damage (COMET-assay) and H2AX phosphorylation were assessed after chloroquine or 3-methyladenine pretreatment and after ATG7 or LC3b knockdown. RESULTS: IR failed to induce autophagy and chloroquine failed to change intrinsic radiosensitivity of cells. Interestingly, 3-methyladenine and ATG7- or LC3b-deficiency sensitized cancer cells to irradiation. Surprisingly, the radiosensitizing effect of 3-methyladenine was also observed in ATG7 and LC3b deficient cells and was associated with attenuated γ-H2AX formation and DNA damage repair. CONCLUSION: Our data demonstrate that the anti-tumor effects of chloroquine are independent of changes in intrinsic radioresistance. Furthermore, ATG7 and LC3b support radioresistance independent of canonical autophagy that involves lysosomal degradation.
Assuntos
Autofagia , Adenina/análogos & derivados , Adenina/farmacologia , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Cloroquina/farmacologia , Reparo do DNA/efeitos dos fármacos , Humanos , Fosforilação , Tolerância a Radiação/genética , Radiação Ionizante , Radiossensibilizantes/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
To efficiently duplicate their genomic content, cells must overcome DNA lesions that interfere with processive DNA replication. These lesions may be removed and repaired, rather than just tolerated, to allow continuity of DNA replication on an undamaged DNA template. However, it is unclear how this is achieved at a molecular level. Here we identify a new replication-associated factor, ZRANB3 (zinc finger, RAN-binding domain containing 3), and propose its role in the repair of replication-blocking lesions. ZRANB3 has a unique structure-specific endonuclease activity, which is coupled to ATP hydrolysis. It cleaves branched DNA structures with unusual polarity, generating an accessible 3'-OH group in the template of the leading strand. Furthermore, ZRANB3 localizes to DNA replication sites and interacts with the components of the replication machinery. It is recruited to damaged replication forks via multiple mechanisms, which involve interactions with PCNA, K63-polyubiquitin chains, and branched DNA structures. Collectively, our data support a role for ZRANB3 in the replication stress response and suggest new insights into how DNA repair is coordinated with DNA replication to maintain genome stability.