Mecanismos moleculares envolvidos em citoproteção e transformação maligna de células-beta pancreáticas / Molecular mechanisms involved in pancreatic beta-cells cytoprotection and malignant transformation

O transplante de ilhotas pancreáticas constitui uma alternativa atraente para o tratamento de diabetes tipo 1 (DM1), contudo, é limitado devido à escassez de doadores de órgãos. O papel da prolactina humana recombinante (rhPRL), que apresenta efeitos benéficos em células-beta, e seu mecanismo de ação foram investigados neste estudo. O número de células apoptóticas diminui significativamente na presença de rhPRL. Essa citoproteção envolveu diminuição da razão BCL2/BAX e inibição de caspase-8, -9 e -3. Este estudo revelou, pela primeira vez, evidência direta do efeito protetor de lactogênios contra apoptose de células-beta humanas. Levando em consideração a relação conhecida entre citocinas e DM1 e observações recentes sugerindo o papel da autofagia no desenvolvimento e prevenção do DM1, foi investigada a conexão entre citocinas (IL-1ß, TNFα e IFN-γ) e autofagia em células-beta. O co-tratamento com citocinas e rapamicina, um indutor de autofagia via inibição de mTOR, não aumentou os níveis de apoptose em células INS-1E. Contudo, exposição a citocinas levou ao aumento nos níveis de autofagossomos e na relação LC3-II/LC3-I, do mesmo modo que o tratamento com rapamicina. O tratamento com citocinas também levou à diminuição dos níveis de mTOR e 4E-BP1 fosforilados. Foi demonstrada aqui, pela primeira vez, uma relação direta entre o tratamento com citocinas e a indução de autofagia em células-beta. Recentemente, surgiram novas evidências mostrando ligação entre a morte de células-beta induzida por citocinas e indução de estresse de retículo endoplasmático. Em nosso modelo, foram observados níveis diminuídos de p-mTOR e aumento da formação de autofagossomos após o tratamento com indutores de estresse de retículo. Este estudo reforça também, resultados prévios sobre a hipótese da função de indutores de estresse de retículo em promover a autofagia. Além disso, o tratamento com rhPRL aumentou os níveis de p-mTOR e levou à diminuição na formação de autofagossomos após exposição a citocinas em células-beta. Estes resultados são relevantes para a caracterização mais aprofundada das funções dos lactogênios nessas células. Sabendo-se da necessidade de células-beta humanas para estudos detalhados em células-beta, nosso grupo gerou linhagens celulares derivadas de insulinomas humanos que secretam hormônios e expressam marcadores com o mesmo padrão de seu tecido original. Estas linhagens foram caracterizadas comparando-as com culturas primárias de células-beta através de eletroforese bidimensional acoplada a espectrometria de massa. Cerca de 1.800 spots foram detectados, sendo que menos de 1% apresentou expressão diferencial. As proteínas superexpressas em ilhotas, como Caldesmon, estão envolvidas em organização do citoesqueleto, influenciando a secreção hormonal. Contrariamente, quase todas as proteínas superexpressas nas células de insulinoma, como MAGE-A2, foram descritas aqui pela primeira vez, podendo estar relacionadas à sobrevivência celular e resistência à quimioterapia. Estes resultados mostram, pela primeira vez, mudanças na expressão de proteínas relacionadas ao fenótipo alterado dos insulinomas, direcionando a pesquisa ao estabelecimento de células-beta humanas bioengenheiradas e ao desenvolvimento de novas estratégias terapêuticas para insulinomas. Coletivamente, os dados obtidos neste estudo estendem o conhecimento molecular envolvido na citoproteção induzida por rhPRL e transformação maligna de células-beta pancreáticas, contribuindo para futuras aplicações na compreensão e no tratamento do DM1

Transplantation of pancreatic islets constitutes an alternative for type 1 diabetes (DM1); however, it is limited by the shortage of organ donors. Here, we investigated the role of recombinant human prolactin (rhPRL), shown to have beneficial effects in beta-cells, and its mechanisms of action. Apoptotic beta-cells were decreased in the presence of rhPRL, with cytoprotection involving an increase of BCL2/BAX ratio and inhibition of caspase-8, -9 and -3. This study provides new direct evidence for a protective effect of lactogens in human beta-cell apoptosis. Taking into account the known relationship between cytokines and DM1 and recent observations suggesting a role for autophagy in the development and prevention of DM1, we investigated the connection between cytokines (IL-1ß, TNF-α and IFN-γ) and autophagy in beta-cells. Co-treatment with cytokines and rapamycin, an inducer of autophagy through inhibition of mTOR, did not increase the apoptosis levels in INS-1E cells. However, exposure to cytokines increased the levels of autophagosome formation and LC3-II/LC3-I ratio. Treatment with cytokines also led to decreased levels of phosphorylated mTOR and 4E-BP1. We demonstrated for the first time, a direct relationship between cytokines treatment and induction of autophagy in beta-cells. Lately, new evidence point to a connection between cytokine-induced beta-cell death and endoplasmic reticulum stress. In our model, we observed that decreased levels of p-mTOR and increased autophagosome formation also ensued after treatment with endoplasmic reticulum stressors. This study also supports the previous hypothesis on the function of ER stressors in inducing autophagy. Furthermore, rhPRL treatment increased the levels of p-mTOR and decreased autophagosome formation after exposure to cytokines in beta-cells. These findings are also relevant for further characterization of lactogens functions in these cells. Considering the demand for human cells for further beta-cells studies, our group generated cell lines derived from human insulinomas which secrete hormones and express markers with the same pattern displayed by their original tissue. We set out to further characterize these lineages by comparing them to primary beta-cells using two-dimensional gel electrophoresis coupled to mass spectrometry. An average of 1,800 spots was detected with less than 1% exhibiting differential expression. Proteins upregulated in islets, such as Caldesmon, are involved in cytoskeletal organization thus influencing hormone secretion. In contrast, almost all proteins upregulated in insulinoma cells, such as MAGE-A2, first described here, could be related to cell survival and resistance to chemotherapy. Our results provide, for the first time, a molecular snapshot of the changes in expression of proteins correlated with the altered phenotype of insulinomas, prompting research towards the establishment of bioengineered human beta-cells, and the development of new therapeutic strategies for insulinomas. Collectively, the data obtained in this study extend the molecular knowledge involved in rhPRL-induced cytoprotection and malignant transformation of pancreatic beta-cells, contributing to future applications for understanding and treatment of DM1

Surgical treatment of pancreatic endocrine tumors in multiple endocrine neoplasia type 1

Surgical approaches to pancreatic endocrine tumors associated with multiple endocrine neoplasia type 1 may differ greatly from those applied to sporadic pancreatic endocrine tumors. Presurgical diagnosis of multiple endocrine neoplasia type 1 is therefore crucial to plan a proper intervention. Of note, hyperparathyroidism/multiple endocrine neoplasia type 1 should be surgically treated before pancreatic endocrine tumors/multiple endocrine neoplasia type 1 resection, apart from insulinoma. Non-functioning pancreatic endocrine tumors/multiple endocrine neoplasia type 1 >1 cm have a high risk of malignancy and should be treated by a pancreatic resection associated with lymphadenectomy. The vast majority of patients with gastrinoma/multiple endocrine neoplasia type 1 present with tumor lesions at the duodenum, so the surgery of choice is subtotal or total pancreatoduodenectomy followed by regional lymphadenectomy. The usual surgical treatment for insulinoma/multiple endocrine neoplasia type 1 is distal pancreatectomy up to the mesenteric vein with or without spleen preservation, associated with enucleation of tumor lesions in the pancreatic head. Surgical procedures for glucagonomas, somatostatinomas, and vipomas/ multiple endocrine neoplasia type 1 are similar to those applied to sporadic pancreatic endocrine tumors. Some of these surgical strategies for pancreatic endocrine tumors/multiple endocrine neoplasia type 1 still remain controversial as to their proper extension and timing. Furthermore, surgical resection of single hepatic metastasis secondary to pancreatic endocrine tumors/multiple endocrine neoplasia type 1 may be curative and even in multiple liver metastases surgical resection is possible. Hepatic trans-arterial chemo-embolization is usually associated with surgical resection. Liver transplantation may be needed for select cases. Finally, pre-surgical clinical and genetic diagnosis of multiple endocrine neoplasia type 1 syndrome and localization of multiple endocrine neoplasia type 1related tumors are crucial for determining the best surgical strategies in each individual case with pancreatic endocrine tumors.

Long-term follow-up of an 8-year-old boy with insulinoma as the first manifestation of a familial form of multiple endocrine neoplasia type 1 / Seguimento de longo prazo em um menino de 8 anos de idade com insulinoma como primeira manifestação de neoplasia endócrina múltipla tipo 1

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant hereditary cancer syndrome characterized mostly by parathyroid, enteropancreatic, and anterior pituitary tumors. We present a case of an 8-year-old boy referred because of hypoglycemic attacks. His diagnosis was pancreatic insulinoma. Paternal grandmother died due to repeated gastroduodenal ulcerations and a paternal aunt presented similar manifestations. At a first evaluation, the father presented only gastric ulceration but subsequently developed hyperparathyroidism and lung carcinoid tumor. During almost 15 years of follow-up, three brothers and the index case presented hyperparathyroidism and hyperprolactinemia. Molecular study showed a G to A substitution in intron 4, at nine nucleotides upstream of the splicing acceptor site, causing a splicing mutation. All affected members of the family have the same mutation. Paternal grandmother and aunt were not studied and the mother does not carry any mutation. MEN1 is a rare condition that requires permanent medical assistance. Early clinical and genetic identification of affected individuals is essential for their own surveillance and also for genetic counseling.

A neoplasia endócrina múltipla tipo 1 (NEM1) é uma doença hereditária autossômica dominante, caracterizada principalmente por tumores de paratireoide, enteropancreáticos e adeno-hipofisários. Apresentamos o caso de um menino com 8 anos encaminhado por crises de hipoglicemia. Seu diagnóstico foi insulinoma pancreático. Sua avó paterna faleceu por úlceras gastroduodenais de repetição e a tia paterna tinha as mesmas manifestações. Na primeira avaliação, o pai apresentou apenas úlcera gástrica, porém com a evolução desenvolveu hiperparatireoidismo e tumor carcinoide pulmonar. Durante cerca de 15 anos de seguimento, os três irmãos e o caso índice desenvolveram hiperparatireoidismo e hiperprolactinemia. O estudo molecular mostrou a substituição G por A no intron 4, a nove nucleotídeos do sítio aceptor de splicing, criando um novo sítio de splicing. Todos os membros da família afetados e estudados tinham a mesma mutação. A NEM1 é uma condição rara que requer assistência médica permanente. As identificações clínicas e genéticas precoces são essenciais para o tratamento e aconselhamento genético.

Perfil de expressão gênica em carcinoma neuroendócrino produtos de insulina: avaliação da agressividade tumoral / Gene expression profiling in insulin-producing neuroendocrine carcinom: tumor aggressiveness evaluation

Insulinomas malignos correspondem entre 5-10per cent de todos os tumores de ilhota. O diagnóstico histopatológico não identifica a malignidade, somente a presença de metástase. Neste estudo foi realizada análise de expressão gênica diferencial em insulinomas com o intuito de identificar genes associados a malignidade. Foram estudados 25 pacientes com insulinoma (2 metástases). Análise de agrupamento hierárquico de alguns genes isolados reuniu os pacientes em grupos com diferentes potenciais malignos, contribuindo para melhor compreensão dos processos carcinogênicos em insulinomas / The gene expression profile associated with benign and malignant insulinomas.Five to 10 per cent insulinomas are malignant, associated with distant metastases or local invasion and histopathological diagnosis cannot identify these tumors as malignant at all. In this study differential gene expression analysis was performed in order to identify genes linked to different aggressive potentials of benign in comparison to malignant insulinomas. Twenty-five patients were studied (two metastases). Hierarchical cluster analysis of expression patterns of some isolated genes put the patients into different groups with progressive malignant potential and that may add new insights into carcinogenesis...

Effect of glucose on the expression of c-myc gene in cultured RINm5F cell

The study was designed to examine the effect of glucose on the expression of c-myc gene in cultured RINm5F cells. After monolayer culture was established in RPMI 1640 media supplemented with 10% fetal calf serum (FCS), the cells were cultured in various concentrations of glucose and 1 or 10% FCS for another 24 hours. A mRNA was extracted from the cultured cells by a single step method, and Northern analysis was done to detect RNA band. A 0.5 kilobase single band was detected as c-myc mRNA. The expression of c-myc gene mRNA was reduced with increased concentration of glucose with 1% FCS. However, supplementation of 10% FCS abolished the effect of glucose on expression of c-myc gene. These findings suggested that glucose in conjunction with other growth promoting factors played an important role in expression of oncogene and cell growth in RINm5F cells.