Neuroblastoma (NB) é a forma mais indiferenciada de tumores neuroblásticos e a principal causa de morte por câncer pediátrico. Alterações epigenéticas interagem em todas as etapas do desenvolvimento do câncer, promovendo a progressão tumoral. A remodelação da cromatina é influenciada pela acetilação de histonas e a metilação de DNA. Acetiltransferases de histona (HATs), desacetilases de histonas (HDAC) e metiltransferase de DNA (DNMTs) são alvos de estratégias terapêuticas em tumores. Os retinoides agem nas vias de diferenciação celular, anti-proliferação e pró-apoptose. Nesse trabalho, é proposto que a combinação desses moduladores epigenéticos e de diferenciação em linhagens de células de NB humano é mais efetiva que os agentes isolados. Os tratamentos induziram mudanças na expressão de marcadores de diferenciação e indiferenciação, como c-Myc, β-3tubulina, NeuN e Bmi1, e alterações morfológicas nas duas linhagens celulares utilizadas, SK-N-BE(2) e SH-SY5Y. Os dados encontrados podem contribuir para uma melhor compreensão dos mecanismos moleculares dos moduladores retinoides e epigenéticos em NB capazes de acrescentar melhorias nas atuais estratégias terapêuticas. / Neuroblastoma (NB) is the most undifferentiated form of neuroblastic tumors and the leading cause of death from pediatric cancer. Epigenetic changes interact at all stages of cancer development, promoting tumor progression. Chromatin remodeling is influenced by histone acetylation and DNA methylation. Histone acetyltransferases (HATs), histone deacetylases (HDAC), and DNA methyltransferase (DNMTs) are targets for therapeutic strategies in cancer. Retinoids act on cell differentiation pathways and display anti-proliferation and pro-apoptotic actions. In the present research we examined the effects of combining epigenetic modulators and a retinoid receptor agonist in human NB cells. The retinoid all trans-retinoic acid (ATRA) combined with inhibitors of either histone deacetylases (HDACs) or DNA methyltransferase was more effective than any drug given alone in impairing the proliferation of SH-SY5Y and SK-N-BE(2) NB cells. In addition, the treatments induced differential changes in the expression of differentiation markers including c-Myc, β-3tubulin, NeuN and Bmi1, and morphological changes in SK-N-BE(2) e SH-SY5Y cell lines. The data contribute to a better understanding of the molecular mechanisms of retinoid modulators and epigenetic in NB able to add improvements in current therapeutic strategies.

A isquemia cerebral causada pela parada cardíaca leva ao desapareciemnto neuronal. studamos os mecanismos de morte celular envolvidos na isquemia in vitro em linhagem de neuroblastoma.O insulto isquêmicao foi reproduzido cultivando as células sem fatores de crescimento, sem glicose e em embiente hipóxico produzido por um sistema de anaerobiose. Os resultados sugerem que a privação de oxigênio, glicose e fatores de cresciemtno do meio de cultura reproduzem o fenômeno semelhante a isquemia. INvestigamos ainda a participação de processo apoptótico e sua modulação térmica. Observams que a hipotermia produz neuroproteção, enquanto a hipertermia agrava o processo de morte celular por apoptose. / Cardiac arrest causes cerebral ischemia and neuronal disappearance. We investigate celular death mechanisms elucidated by a model of ischemia in neuroblastoma cell line. The ischemic insult was reproduced by deprivation of growth factors and glucose in a hypoxic environment produced by an anaerobiosis system. Our results validate the experimental model and revel the participation of an apoptotic process in the celular loss induced by ischemia. We also demonstrated that hypothermia can be used as a neuroprotector agent whereas hyperthermia aggavates celular damage.

No description available.

published_or_final_version / abstract / Surgery / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

The molecular mechanisms of neuroblastoma tumorigenesis include arrested neuritic differentiation, making an attractive strategy for treatments which promote differentiation of this childhood disease. The N-Myc oncoprotein has an established role in neuroblastoma tumorigenesis, although the exact mechanism is poorly defined. Treatment side-effects are extensive in treatment of children receiving conventional chemo-radiotherapy for neuroblastoma. Retinoids have a low side-effect profile and are used in children with neuroblastoma at the point of minimal residual disease. Retinoids can simultaneously combat N-Myc effects, induce differentiation, growth inhibition, and, cause cancer cell death. Retinoid Acid Receptor beta (RARβ) is an important mediator of the retinoid anti-cancer effect in neuroblastoma cells. I have found that perinatal environmental factors such as hypoxia and nutrient reduction can contribute to neuroblastoma initiation by conferring resistance to a subsequent physiologic death stimulus of NGF withdrawal. We also concluded that transient N-myc expression perinatally may play a central role in neuroblastoma initiation, for it was instrumental in rendering death resistance towards a range of different stress stimuli. We hypothesise that the effects of transient perinatal stress on ganglia cells may be mimicked by the transient N-Myc expression reproduced in the N-Myc mouse model. I also found that five known N-Myc transcriptional target genes (ODC1, MCM7, MRP1, hTERT and α-Prothymosin) are over-expressed in perinatal paravertebral ganglia fromJhe N-Myc mouse model. I also identified Myc Box" domain of the N-Myc protein as being necessary for resistance to NGF withdrawal. I also sought to better understand the mechanisms of retinoid differentiation treatment for disease. We sought to define the role of a novel retinoid co-regulator, Estrogen-Responsive B Box Protein (EBBP), in the propagation of the retinoid differentiation anti-cancer signal. We hypothesized that EBBP over-expression can restore retinoid sensitivity in vitro in retinoid-resistant cells, and, that retinoid resistance can be overcome by EBBP over-expression in combination with all-trans-retinoic acid (atRA), a demethylating agent or a histone deacetylase (HDAC) inhibitor. In order to address these hypotheses, we used a panel of retinoid-resistant lung and breast cancer cell lines transiently transfected with either a control plasmid or EBBP plasmid. We then employed Real Time PCR, BrdU, Alamar Blue and cell death detection assays to investigate the effect of EBBP over-expression on RARβ transcription, cell proliferation, cell viability, apoptosis and necrosis in the presence, or absence, of atRA, a demethylating agent (5-Aza), or a HDAC inhibitor (Trichostatin A, TSA). We found that EBBP over-expression can increase sensitivity to TSA and all trans retinoic acid (atRA) treatment, reduce cell proliferation and viability, trigger cell death, and, more importantly induce RARβ and Retinoic Acid Hydroxylase (CYP26A) transcription ,in RA-resistant cancer cells. We conclude that while EBBP can augment some of the effects of existing treatment agents, it may resent a novel target for differentiation therapy in retinoid-resistant cancer cells.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Hawaii at Manoa, 2003. / Includes bibliographical references (leaves 122-177).

Thesis (Ph. D.)--University of Hawaii at Manoa, 2003. / Includes bibliographical references (leaves 122-177). Also available by subscription via World Wide Web.