O desenvolvimento de moléculas radiomarcadas com alta especificidade para um órgão ou tumor tem contribuído para a obtenção de um diagnóstico de precisão em medicina nuclear.Um caso particular são os peptídeos radiomarcados para a localização de tumores neuroendócrinos como os derivados sintéticos da somatostatina.Atualmente, o DTPA-octreotideo-111In é o radiofármaco mais utilizado com o propósito de visualizar tumores que expressem receptores para somatostatina. Contudo, o uso do indio-111 como radionuclídeo oferece limitações em relação a sua disponibilidade (produto de ciclotron), suas características físicas como meia-vida (67 horas) e emissor de fótons de média energia (171 keV e 245 keV) que não favorecem a obtenção de imagens tipo SPECT (Single Photon Emission Computed Tomography). As propriedades físicas favoráveis do tecnécio-99m (99mTc) fazem dele o radioisótopo mais adequado para substituir o indio-111 (111In) na marcação desses peptídeos. Este trabalho avaliou a preparação e marcação do reagente liofilizado HYNIC-Tyr3-octreotato (HYNIC-octreotato) com 99mTc, baseado em metodologia descrita na literatura, utilizando tricina e EDDA (ácido etilenodiaminadiacetico) como coligantes. Foram estudados os parâmetros de marcação (tempo de incubação, temperatura, volume e atividade do pertecnetato de sódio) e estabilidade do liofilizado. Adicionalmente, estudou-se a influência de pré-congelamento com nitrogênio (N2) líquido na estabilidade do liofilizado, bem como a influência de manitol na pureza radioquímica e biodistribuição do complexo. Os estudos de estabilidade revelaram que o método de liofilização utilizado, empregando o pré-congelamento com nitrogênio líquido possibilitou a obtenção de um reagente liofilizado com estabilidade de 4 meses quando armazenado sob refrigeração. A estabilidade do reagente liofilizado obtido sem pré-congelamento com nitrogênio líquido foi semelhante à obtida com o pré-congelamento.Os estudos de marcações determinaram as melhores condições de marcação, para as quais se obteve pureza radioquímica maior que 90%.A presença de manitol na formulação não influenciou na formação do complexo HYNIC-Octreotato-99mTc, conforme avaliação realizada por CLAE e estudos cintilográficos de distribuição do composto em coelhos.Estudos de biodistribuição invasivos realizados em camundongos Nude com tumor (células AR42J de tumor pancreático) e camundongos Swiss normais, bem como estudos cintilográficos realizados em coelhos e camundongos Nude revelaram cinética de distribuição rápida, acúmulo renal e captação tumoral significativa do peptídeo radiomarcado. Os resultados dos estudos de marcação com 99mTc, produção de reagente liofilizado e biodistribuição sugerem que o radiofármaco HYNIC-octreotato-99mTc apresenta potencial para aplicação em diagnóstico de tumores neuroendócrinos em medicina nuclear. / The development of radiolabeled molecules with high specificity for an organ or tumor has been contributed to the precise diagnostic in nuclear medicine. Somatostatin labeled derivatives constitutes a particular example of labeled peptide applied in the localization of neuroendocrine tumors. Nowadays, the 111In-DTPA-octreotideo is the radiopharmaceutical applied in diagnostic procedures for the visualization of tumors with high expression of somatostatin receptors. However, the 111-indium is a radionuclide that presents some limitations related to availability (cyclotron production), half-life (67 hours) and the emission of medium energy photons (171 keV e 245 keV), not favorable to the acquisition of images in SPECT (Single Photon Emission Computed Tomography). The favorable physical properties of the 99m-technetium (99mTc) make this radionuclide the more favorable to substitute the 111-indium on peptide labeling procedures. This work studied the preparation and labeling of a lyophilized kit of HYNIC-Tyr3-octreotate (HYNIC-octreotate) with 99mTc, base on previously described procedures and using tricine and EDDA (ethylendiaminediacetic acid) as coligands. It was studied the labeling parameters (incubation time, temperature, volume and perthecnetate activity) and the stability of the lyophilized preparation. Additionally, it was studied the influence of the pre-freezing using liquid nitrogen in the stability of the lyophilized preparation, as well as the influence of manitol in the labeling yield and biological distribution of the complex. The stability studies showed that the lyophilization using liquid nitrogen pre-freezing resulted in a lyophilized preparation with stability over 4 month when stored under refrigeration. The stability of the lyophilized preparation obtained without liquid nitrogen pre-freezing was similar.The labeling studies determined the best labeling conditions, resulting in a radiochemical yield superior than 90%. The use of manitol in the formulation did not influence the formation of the complex 99mTc-HYNIC-Octreotate, as evidenced in HPLC and in the scintigraphic studies of the complex biodistribution in rabbits. Invasive biodistribution studies using xenographed Nude mice (pancreatic tumor cells AR42J) and healthy Swiss mice, and scintigraphic studies in rabbits showed the fast kinetic distribution, renal uptake and significative tumoral uptake of the labeled peptide. The results of labeling studies with 99mTc, the production of the lyophilized kit and the biodistribution studies suggest that the 99mTc-HYNIC-Octreotate is a potential radiopharmaceutical to be applied in the diagnostic of neuroendocrine tumors in nuclear medicine.

Os insulinomas são os mais comuns neoplasmas endócrinos pancreáticos, constituindo cerca de 17% de todos os tumores neuroendócrinos do trato digestivo. São tumores raros, que tem, como principal manifestação clínica, a hipoglicemia, a qual é ocasionada por secreção exagerada de insulina pelo tumor. Devido ao fato de serem tumores raros, o conhecimento das mudanças genéticas associadas à iniciação e progressão desses tumores é muito limitado. Em função disto, o objetivo deste trabalho é a identificação de genes diferencialmente expressos em insulinomas benignos humanos, visando o melhor entendimento dos mecanismos moleculares do processo tumorigênico dos insulinomas e a descoberta de novos alvos moleculares para terapia. Utilizando-se a plataforma de \"bioarrays\" CodeLink foram identificados 354 genes mais expressos nos insulinomas benignos, sendo que 16% estavam envolvidos em proliferação. Dentre estes genes foram escolhidos 6 genes para validação por \"Real-Time PCR\", onde os genes SPARCL1, PRSS11 STAT4, ECRG4, ASCL1 confirmaram sua expressão diferencial nos tumores, porém a diferença do gene IGFALS não foi estatisticamente significativa. Através da técnica \"Representational Difference Analysis\", isolou-se o clone FLJ13072, como super-expresso nos insulinomas benignos quando comparado à ilhotas normais, sendo que a seqüência protéica putativa deste gene apresenta um domínio conservado de helicase, podendo estar envolvido em eventos de transcrição, tradução, reparo de DNA e remodelamento de cromatina. Uma das dificuldades encontradas no estudo dos insulinomas é a falta de linhagens celulares humanas. Por esta razão, iniciou-se o estabelecimento de culturas primárias e precoces de insulinomas humanos visando sua utilização como modelos celulares para futuros estudos funcionais dos genes identificados. / Insulinomas are the most common pancreatic endocrine neoplasms, comprising around 17% of all neuroendocrine tumors of the digestive tract. These rare tumors have hypoglycemia as the main clinical manifestation, caused by over secretion of insulin by the tumor. Based on that, the objective of this work is the identification of differentially expressed genes in human benign insulinomas, aiming at the better understanding of the molecular mechanisms of their tumorigenic process and the discovery of new molecular targets for therapeutics. Using the CodeLink bioarrays platform (GE Healthcare) 354 genes upregulated in human benign insulinomas were identified, among which, 16% are involved in cell proliferation. From these genes, 6 were chosen for validation by Real Time PCR, where SPARCL1, PRSS11, STAT4, ECRG4 and ASCL1 were shown to be upregulated in all benign tumors, however the expression difference of IGFALS gene were not statistically significant. Using the RDA (Representational Difference Analysis) methodology, the unknown gene FLJ13072 was shown to be upregulated in benign isulinomas when compared to normal pancreatic islets. The putative protein product from this gene has an helicase domain, being possibly involved in processes like transcription, translation, DNA repair and chromatin remodeling. An important drawback for the study of insulinomas is the lack of human cell lines. Because of that, the establishment of early primary cultures of human insulinomas was initiated, aiming at its use as a cell model for future functional studies of the genes identified.

IFN-α has been applied in medical treatment of various neuroendocrine (NE) tumors, either alone or combination with somatostatin analogues. They can improve clinical symptoms in 50-70% of patients but a significant tumor reduction is only observed in 5-15% patients. Vasostatin (vaso) is believed to be an angiogenic inhibitor. The aim of this study is to evaluate the feasibility to use IFN-α and vasostatin gene therapy in NE tumors. We constructed plasmid vectors carrying human IFN-α2 (hIFN-α2) gene and human vasostatin gene, which were transfected into BON I cell to obtain stable gene-expressing cell lines. We found that in animal tumor model and cell experiments gene transfer of vasostatin caused a faster cell growth and tumor development via down-regulation of the tumor suppressor gene and p27. Cell adhesion, spreading, migration and invasion ability were increased in vaso-expressing BON I cells. Transfecting chicken vinculin could reverse the malignant behavior and restored expression of tumor suppressor genes. Moreover, vinculin knockdown could result in a faster cell growth and an increased colony formation. Condition medium taken from hIFN-α2-expressing BON I cells showed significant antiproliferative effects both on the NE tumor cells, BON I and LCC18, and the endothelial cells, PAE. It also suppressed cell adhesion and cell invasion and inhibited angiogenesis on CAM assay. Mice implanted with a mixture of WT BON cells and hIFN-α2-expressing BON cells (1:1) demonstrated significantly lower tumor incidence and longer tumor doubling time. Furthermore, long-acting IFN-α2b (PEGIntron®) demonstrated a better anti-tumor effect in contrast with IFN-α2b (IntronA®). Intratumoral injection of hIFN-α2 plasmids significantly inhibited NE tumor growth and caused tumor regression. We concluded that gene transfer of vasostatin into BON I cells might cause an enhanced malignant tumor behavior. Therefore, vasostatin therapy can not be recommended for patients with NE tumors. Vinculin might play an important role in NE tumor development and growth regulation. Gene therapy by using plasmid DNA carrying hIFN-α2 gene is feasible and promising in NE tumors.

Adenovirus serotype 5 (Ad5) is widely used as an oncolytic agent for cancer therapy. However, its infectivity is highly dependent on the expression level of coxsackievirus-adenovirus receptor (CAR) on the surface of tumor cells. We engineered Ad5 virus with the protein transduction domain (PTD) from the HIV-1 Tat protein (Tat-PTD) inserted in the hypervariable region 5 (HVR5) of the hexon protein in the virus capsid. Tat-PTD-modified Ad5 shows a dramatically increased transduction level of CAR-negative cells and bypassed fiber-mediated transduction. It also overcomes the fiber-masking problem, which is caused by release of excess fiber proteins from infected cells. To achieve specific viral replication in neuroblastoma and neuroendocrine tumor cells, we identified the secretogranin III (SCG3) promoter and constructed an adenovirus Ad5PTD(ASH1-SCG3-E1A) wherein E1A gene expression is controlled by the SCG3 promoter and the achaete-scute complex homolog 1 (ASH1) enhancer. This virus shows selective and efficient killing of neuroblastoma cell lines in vitro, and delays human neuroblastoma xenograft tumor growth on nude mice. To further enhance the viral oncolytic efficacy, we also switched the fiber 5 to fiber 35 to generate Ad5PTDf35. This vector shows dramatically increased transduction capacity of primary human cell cultures including hematopoietic cells and their derivatives, pancreatic islets and exocrine cells, mesenchymal stem cells and primary tumor cells including primary cancer initiating cells. Ad5PTDf35-based adenovirus could be a useful platform for gene delivery and oncolytic virus development. Viral oncolysis alone cannot completely eradicate tumors. Therefore, we further armed the Ad5PTDf35-D24 virus with a secreted form of Helicobacter pylori Neutrophil Activating Protein (HP-NAP). Expression of HP-NAP recruits neutrophils to the site of infection, activates an innate immune response against tumor cells and provokes a Th1-type adaptive immune response. Established tumor on nude mice could be completely eradicated in some cases after treatment with this virus and the survival of mice was significantly prolonged.

Acute brain cell swelling (cytotoxic edema) can occur in the first minutes of stroke, presumably as a result of brain cells taking up water. In extreme hypo-osmotic situations such as excessive water-loading by patients, uptake by brain cells can expand the brain, causing seizures. But is ischemic brain cell swelling the same as hypo-osmotic swelling? Water can passively diffuse across the plasma membrane. However the presence of water channels termed aquaporins (AQP) facilitates passive water diffusion by 10-100 times. Unlike astrocytes, there is no evidence of water channels on neuronal plasma membrane. However, there is still much debate about which cells (neurons or astrocytes) swell during over-hydration or during stroke and if neurons and astrocytes can volume-regulate during osmotic stress. The purpose of this study was to examine and compare the volume responses of PyNs and magnocellular neuroendocrine cells (MNCs) to acute osmotic challenge and to OGD. We examined MNCs because they are intrinsically osmosensitive to small changes (2-3 mOsm) of plasma osmolality. We also examined if the same neurons behave similarly in brain slices or when dissociated and if they respond differently to acute osmotic stress and stroke-like stress. Our results indicate that during acute osmotic stress (±40 mOsm) half of dissociated PyNs and MNCs tended to show appropriate responses. MNCs in brain slices showed similar responses to when they were dissociated, while brain slice PyNs were less responsive than when dissociated. Exposure to OGD resulted in obvious differences between the two types of in vitro preparations. Dissociated PyNs and MNCs showed no consistency in their volume responses to 10 minutes of OGD. Dissociated neurons swelled, shrunk or were unchanged in about equal numbers. In contrast, brain slice PyNs underwent profound swelling whereas, brain slice MNCs showed minor volume decreases. We conclude that about half of our dissociated neurons were too variable and unpredictable in their osmotic volume responses to be useful for osmotic studies. They also were too resistant to stroke-like stress to be good models for ischemia. Brain slice neurons were similar in their osmotic responses to dissociated neurons but proved to have consistent and predictable responses to stroke-like stress. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2011-02-07 17:55:08.333

Small intestinal neuroendocrine tumors (SI-NETs) originate from serotonin-producing enterochromaffin (EC) cells in the intestinal mucosa. Somatostatin analogs (SSAs) are mainly used to control hormonal secretion and tumor growth. However, the molecular mechanisms leading to the control of SI-NETs are unknown. Although microRNAs (miRNAs) are post transcriptional regulators deeply studied in many cancers, are not well-defined in SI-NETs. We adopted a two-pronged strategy to investigate SSAs and miRNAs: first, to provide novel insights into how SSAs control NET cells, and second, to identify an exclusive SI-NET miRNA expression, and investigate the biological functions of miRNA targets. To accomplish the first aim, we treated CNDT2.5 cells with octreotide for 16 months. Affymetrix microarray was performed to study gene variation of CNDT2.5 cells in the presence or absence of octreotide. The study revealed that octreotide induces six genes, ANXA1, ARHGAP18, EMP1, GDF15, TGFBR2 and TNFSF15. To accomplish the second aim, SI-NET tissue specimens were used to run genome-wide Affymetrix miRNA arrays. The expression of five miRNAs (miR-96, -182, -183, -196a and -200a) was significantly upregulated in laser capture microdissected (LCM) tumor cells versus LCM normal EC cells, whereas the expression of four miRNAs (miR-31, -129-5p, -133a and -215) was significantly downregulated in LCM tumor cells. We also detected nine tissue miRNAs in serum samples, showing that the expression of five miRNAs is significantly increased in SSA treated patients versus untreated patients. Conversely, SSAs do not change miRNA expression of four low expressed miRNAs. Silencing miR-196a expression was used to investigate functional activities in NET cells. This experimental approach showed that four miR-196a target genes, HOXA9, HOXB7, LRP4 and RSPO2, are significantly upregulated in silenced miR-196a NET cells. In conclusion, ANXA1, ARHGAP18, EMP1, GDF15, TGFBR2 and TNFSF15 genes might regulate cell growth and differentiation in NET cells, and play a role in an innovative octreotide signaling pathway. The global SI-NET miRNA profiling revealed that nine selected miRNAs might be involved in tumorigenesis, and play a potential role as novel markers for follow-up. Indeed, silencing miR-196a demonstrated that HOXA9, HOXB7, LRP4 and RSPO2 genes are upregulated at both transcriptional and translational levels.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2008. / Härtill 4 uppsatser.

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 25, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: Dr. Silvia Jurisson and Dr. Mike Lewis. Vita. Includes bibliographical references.

Thesis (Ph. D. in Cancer Biology)--Vanderbilt University, Aug. 2007. / Title from title screen. Includes bibliographical references.

Pancreatic neuroendocrine tumours (PNETs) are the most common neuroendocrine tumours diagnosed in humans and dogs. Due to the highly heterogeneous nature of these tumours, definitive data are still lacking over the molecular mechanisms involved in their cancerous behaviour. This study focused on insulinoma (INS), as it is the most commonly diagnosed PNET in human and veterinary oncology. INS is an insulin-producing tumour that causes a hypoglycaemic syndrome related to the excessive insulin production. In humans, it is often a small benign neoplasm readily curable by surgical resection whereas, in dogs, INS is often malignant. Despite current treatment modalities, malignant canine and human INS have a poor prognosis as patients tend to develop metastases in liver and lymph nodes that do not respond to current therapies. From a comparative oncology perspective, the close resemblance of canine and human malignant INS makes canine INS an interesting study model for human INS. Cancer stem cells (CSCs) are critical for the engraftment and chemoresistance of many tumours. Although CSCs have been isolated from a range of solid tumours, a comprehensive characterisation of INS CSCs has not yet been reported. In this study, it was confirmed that INS CSCs can be enriched and are potential targets for novel INS therapies. Highly invasive and tumourigenic human and canine INS CSCs were successfully isolated and exhibited greater resistance to chemotherapy, which may play a significant role in the poor prognosis of this disease. To date, the mechanisms by which tumours spread and the clinical causes of chemoresistance remain only partially understood. Here, RNA-sequencing analysis was performed over a small set of canine INS tumour samples in order to identify mechanisms involved in INS carcinogenesis through different stages of the disease. Preliminary data showed that distinct gene profiles characterised early and late stage of canine INS. Interestingly, differential gene expression and gene pathways analysis, highlighted that sets of genes involved in pancreatic embryogenesis and insulin secretion were overexpressed in canine primary INS lesions compared with normal pancreas. The Notch pathway is fundamental in pancreatic embryogenesis and it has been previously associated with carcinogenesis of neuroendocrine tumours and with the CSC phenotype. Protein analysis showed that the Notch pathway is activated in both human and canine INS CSCs, particularly when treated with chemotherapy, indicating that the Notch pathway may be involved in chemoresistance. Additionally, it was demonstrated that inhibition of the Notch pathway decreased INS CSCs' survival and chemoresistance, both in vitro and in vivo. These findings provide preclinical evidence that anti-Notch therapy may improve outcomes for patients with malignant INS.