The formation of neurospheres was important in demonstrating that neurogenesis in the adult brain may be fuelled by a stem cell population. The olfactory mucosa is another site of neurogenesis which, in humans, has been observed to contain a stem cell population through the formation of neurospheres (Murrell et al., 2005). Stem cells can be defined as cells capable of self-renewal and multipotency. The aim of this study was to investigate the potential of rat olfactory stem cells growing as neurospheres. The hypothesis is that the rat olfactory mucosa contains a 'true' stem cell population that can be cultured as neurospheres and that will demonstrate multipotency by differentiating into 'non-olfactory' cell types and possess the capacity for self-renewal, if provided with the appropriate environmental niche. Here it was found that adult rat olfactory mucosa is capable of generating neurospheres when cultured in EGF and bFGF. Evidence of self-renewal was provided by the formation of six generations of neurospheres, the formation of neurospheres from single cells and the expression of markers associated with self-renewal by neurosphere cells. The multipotency of olfactory neurosphere cells was demonstrated through manipulation of the stem cell niche. In defined culture conditions, extracellular matrix molecules and growth factors were able to induce the differentiation of neurosphere cells down the dopaminergic lineage pathway. When co-cultured with differentiating cells, neonatal myoblasts and 3T3-L1 cells, olfactory neurosphere cells were able to differentiate and incorporate into a skeletal muscle myotube and differentiate into adipocytes, respectively. In conclusion it was found that the adult rat olfactory mucosa is capable of generating neurospheres. When presented with an appropriate niche neurosphere cells are able to self-renew and demonstrate multipotency.

No description available.

Introdução: O glioblastoma multiforme é a neoplasia maligna primária mais prevalente do sistema nervoso central. Enquanto apresenta aumento em sua incidência, mantém a perspectiva de sobrevida global aproximada de 14 meses. Objetivos: O presente estudo objetiva avaliar a expressão dos genes EGFR, PTEN, MGMT e IDH1/2 e dos microRNAs miR-181b, miR-145, miR-149 e miR-128a em neuroesferas (NE) e células aderidas (CA), a partir das linhagens celulares (T98G e U343) submetidas ao tratamento com temozolomida (TMZ) e com radiação ionizante (RI) isolados e em associação (TMZ+RI). Material e métodos: As linhagens celulares T98G e U343 foram tratadas com TMZ, RI e TMZ+RI. A verificação da expressão dos genes e miRNAs foi realizada utilizando o método de PCR em tempo real. Resultados: Observamos: a) o aumento na expressão do IDH1 após RI (4-fold) e TMZ+RI (5-fold) nas NE (T98G); o aumento na expressão do IDH2 (20-fold) nas NE (T98G), após TMZ, e do IDH2 (3-fold) nas CA (U343) submetidas à TMZ+RI; b) a redução na expressão do EGFR após TMZ; o aumento dessa expressão nas NE (T98G), após RI (2-fold) e TMZ+RI (3-fold), e a sua diminuição nas CA (U343) submetidas à TMZ e TMZ+RI; c) a redução na expressão PTEN nas NE (T98G), após TMZ e RI, e sua expressão nas NE (3-fold) frente à TMZ+RI; d) aumento na expressão de MGMT nas NE (T98G) nos grupos RI (10-fold) e TMZ+RI (25-fold). Quanto a expressão de miRNAs, foram identificados os seguintes resultados: a) as CA (U343) expressaram: miR-181b após TMZ (60-fold), RI (20-fold) e TMZ+RI (40- fold); e miR-128a após TMZ (500-fold), RI (200-fold) e TMZ+RI (600-fold); b) as NE (T98G) após TMZ+RI expressaram: miR-181b (30-fold), miR-149 (40-fold), miR-145 (300-fold) e miR-128a (40-fold); c) as NE (U343) após RI hiperexpressaram miR-149 e miR-145 (ambos em 4000-fold). Conclusão: A RI apresentou-se como fator independente e determinante para a radiorresistência das NE, entretanto não observamos ação de complementariedade de regulação dos oncomiRs observados sobre a expressão dos genes estudados. Esta é a primeira análise na literatura que correlaciona a expressão de genes e de miRNAs através das intervenções TMZ, RI e TMZ+RI sobre células aderidas e neurosferas. / Introduction: Glioblastoma multiforme is the most prevalent primary malignant neoplasm of the central nervous system. It has increased its incidence, while the overall survival remains over 14 months. Objectives: This study aims to evaluate the expression of the following genes EGFR, PTEN, MGMT and IDH1/2 and microRNAs miR-181b, miR-145, miR-149 and miR-128a in adhered cells (AC) and neurospheres (NS) from cell lines (T98G and U343) which were submitted to temozolomide (TMZ) and ionizing radiation (IR), isolated and associated (TMZ + IR). Methods: The cell lines T98G and U343 were treated with TMZ, IR and TMZ+IR. The analysis of gene expression and miRNAs was performed using the PCR in real time. Results: This study demonstrated: a) an improvement in the expression of IDH1 after IR (4-fold) and TMZ + IR (5-fold) in the NS (T98G); increased expression of IDH2 (20-fold) in the NS (T98G) after TMZ and IDH2 (3-fold) in AC (U343) submitted to TMZ + IR; b) reduction in EGFR expression after TMZ; increase this expression in NS (T98G) after IR (2-fold) and TMZ + IR (3-fold), and a decrease in AC (U343) submitted to TMZ and TMZ + IR; c) reduction in PTEN expression in NS (T98G) after TMZ and IR, and its improvement in NS (3-fold) when compared to TMZ + IR; d) increase in the expression of MGMT in NS (T98G) in IR groups (10-fold) and TMZ + IR (25-fold). It was also identified the expression of miRNAs results as: a) AC (U343) expressed more miR-181b after TMZ (60-fold), IR (20-fold) and TMZ + IR (40-fold); and miR- 128a improved after TMZ (500-fold), IR (200-fold) and TMZ + IR (600-fold); b) NS (T98G) after TMZ + IR expressed: miR-181b (30-fold); miR-149 (40-fold); miR-145 (300-fold) and miR-128a (40-fold); c) NS (U343) after IR huge expressed miR-149 and miR-145 (both in 4000-fold). Conclusion: IR was an independent and determining radio resistance factor in NS. However, we observed no complementarity action of oncomiRs regulation. This is the first study in the literature correlating gene expression, and miRNAs through TMZ, IR and IR + TMZ interventions in AC and NS.

Le glioblastome représente le sous-type de tumeur cérébrale le plus fréquent et le plus agressif. Malgré une meilleure compréhension de la maladie ainsi que l'émergence de nouvelles cibles, voire de nouveaux outils thérapeutiques, son pronostic reste inchangé. En effet, l'échec de l'extrapolation des résultats vers la clinique met en exergue la nature complexe de la maladie et la dimension décisive des modèles animaux adéquats et prédictifs dans l'étude des nouvelles thérapies. Et pour cause, un modèle animal idéal doit pouvoir reproduire les caractéristiques histo-pahologiques, génétiques et diagnostics de la pathologie humaine. Il doit avoir également une survie suffisante pour permettre la mise en place et l'évaluation de nouveaux traitements. Au cours de ce travail, nous avons développé un nouveau modèle de tumeur orthotopique chez la souris à partir de cellules de glioblastome humain cultivées en neurosphères. D'une façon similaire aux protocoles cliniques de neuro-imagerie, les techniques classiques d'IRM et les critères radiographiques ont été utilisés afin d'étudier la croissance tumorale de ce modèle ainsi que l'évaluation de sa réponse au traitement à base de temozolomide. Les observations par imagerie ont été complétées et/ou confirmées par examen histologique ainsi que par l'étude du transcriptome. Comme en clinique, ce nouveau modèle orthotopique présente une tumeur invasive et nécrotique, une résistance au temozolomide ainsi que des signatures moléculaires associées aux observations histologiques. De plus, ce modèle tumoral est caractérisé par une dynamique de signalisation promouvant l'invasion, la migration et la résistance à l'apoptose à l'origine de sa survie post-thérapeutique. Ainsi, ce modèle préclinique mime, au plus près, les caractéristiques de la pathologie humaine avec une médiane de survie des animaux de 82 jours, ce qui le rend pertinent pour l'évaluation préclinique des nouvelles stratégies thérapeutiques. / Glioblastoma is the most common and aggressive subtype of brain tumors. Despite a better understanding of the disease and also the emergence of new therapeutic targets and strategies, the prognosis of patients remains unchanged. The failure to extrapolate preclinical results to the clinics highlights the complex nature of the disease and the importance of appropriate and predictive animal models for the study of new therapies. A pertinent animal model should be able to reproduce the characteristic of the human pathology in terms of disease development pattern, histological and transcriptomic specification, drug failure as well as diagnostic features. In this work, we developed a novel orthotopic mouse model derived from human glioblastoma spheres. Like in clinics, conventional MRI techniques and radiographic criteria were used to characterize tumor growth and treatment response to temozolomide. MRI findings have been completed and/or confirmed by histological examination and transcriptomic studies. Like clinically encountered tumors, this new orthotopic tumor model presents an infiltrating growth pattern, resistance to temozolomide and a molecular signature associated with histological features. In addition, this tumor model is characterized by a dynamic signaling pathway, which promotes cell invasion and migration as well as resistance to apoptosis and consequently to treatment. Thus, this preclinical model mimics clinical features of human glioblastoma and has a median host survival time of 82 days, which would be relevant in the assessment of preclinical therapies.

Introdução: O glioblastoma multiforme é a neoplasia maligna primária mais prevalente do sistema nervoso central. Enquanto apresenta aumento em sua incidência, mantém a perspectiva de sobrevida global aproximada de 14 meses. Objetivos: O presente estudo objetiva avaliar a expressão dos genes EGFR, PTEN, MGMT e IDH1/2 e dos microRNAs miR-181b, miR-145, miR-149 e miR-128a em neuroesferas (NE) e células aderidas (CA), a partir das linhagens celulares (T98G e U343) submetidas ao tratamento com temozolomida (TMZ) e com radiação ionizante (RI) isolados e em associação (TMZ+RI). Material e métodos: As linhagens celulares T98G e U343 foram tratadas com TMZ, RI e TMZ+RI. A verificação da expressão dos genes e miRNAs foi realizada utilizando o método de PCR em tempo real. Resultados: Observamos: a) o aumento na expressão do IDH1 após RI (4-fold) e TMZ+RI (5-fold) nas NE (T98G); o aumento na expressão do IDH2 (20-fold) nas NE (T98G), após TMZ, e do IDH2 (3-fold) nas CA (U343) submetidas à TMZ+RI; b) a redução na expressão do EGFR após TMZ; o aumento dessa expressão nas NE (T98G), após RI (2-fold) e TMZ+RI (3-fold), e a sua diminuição nas CA (U343) submetidas à TMZ e TMZ+RI; c) a redução na expressão PTEN nas NE (T98G), após TMZ e RI, e sua expressão nas NE (3-fold) frente à TMZ+RI; d) aumento na expressão de MGMT nas NE (T98G) nos grupos RI (10-fold) e TMZ+RI (25-fold). Quanto a expressão de miRNAs, foram identificados os seguintes resultados: a) as CA (U343) expressaram: miR-181b após TMZ (60-fold), RI (20-fold) e TMZ+RI (40- fold); e miR-128a após TMZ (500-fold), RI (200-fold) e TMZ+RI (600-fold); b) as NE (T98G) após TMZ+RI expressaram: miR-181b (30-fold), miR-149 (40-fold), miR-145 (300-fold) e miR-128a (40-fold); c) as NE (U343) após RI hiperexpressaram miR-149 e miR-145 (ambos em 4000-fold). Conclusão: A RI apresentou-se como fator independente e determinante para a radiorresistência das NE, entretanto não observamos ação de complementariedade de regulação dos oncomiRs observados sobre a expressão dos genes estudados. Esta é a primeira análise na literatura que correlaciona a expressão de genes e de miRNAs através das intervenções TMZ, RI e TMZ+RI sobre células aderidas e neurosferas. / Introduction: Glioblastoma multiforme is the most prevalent primary malignant neoplasm of the central nervous system. It has increased its incidence, while the overall survival remains over 14 months. Objectives: This study aims to evaluate the expression of the following genes EGFR, PTEN, MGMT and IDH1/2 and microRNAs miR-181b, miR-145, miR-149 and miR-128a in adhered cells (AC) and neurospheres (NS) from cell lines (T98G and U343) which were submitted to temozolomide (TMZ) and ionizing radiation (IR), isolated and associated (TMZ + IR). Methods: The cell lines T98G and U343 were treated with TMZ, IR and TMZ+IR. The analysis of gene expression and miRNAs was performed using the PCR in real time. Results: This study demonstrated: a) an improvement in the expression of IDH1 after IR (4-fold) and TMZ + IR (5-fold) in the NS (T98G); increased expression of IDH2 (20-fold) in the NS (T98G) after TMZ and IDH2 (3-fold) in AC (U343) submitted to TMZ + IR; b) reduction in EGFR expression after TMZ; increase this expression in NS (T98G) after IR (2-fold) and TMZ + IR (3-fold), and a decrease in AC (U343) submitted to TMZ and TMZ + IR; c) reduction in PTEN expression in NS (T98G) after TMZ and IR, and its improvement in NS (3-fold) when compared to TMZ + IR; d) increase in the expression of MGMT in NS (T98G) in IR groups (10-fold) and TMZ + IR (25-fold). It was also identified the expression of miRNAs results as: a) AC (U343) expressed more miR-181b after TMZ (60-fold), IR (20-fold) and TMZ + IR (40-fold); and miR- 128a improved after TMZ (500-fold), IR (200-fold) and TMZ + IR (600-fold); b) NS (T98G) after TMZ + IR expressed: miR-181b (30-fold); miR-149 (40-fold); miR-145 (300-fold) and miR-128a (40-fold); c) NS (U343) after IR huge expressed miR-149 and miR-145 (both in 4000-fold). Conclusion: IR was an independent and determining radio resistance factor in NS. However, we observed no complementarity action of oncomiRs regulation. This is the first study in the literature correlating gene expression, and miRNAs through TMZ, IR and IR + TMZ interventions in AC and NS.

Glioblastoma Multiforme (GBM) is the most common lethal brain tumour associated with dismal survival rate. GBM is considered to be an incurable malignancy as these tumours evade all intricate attempts of therapy and no contemporary chemotherapeutic regimen is effective. Although the existence of cancer stem cells (CSCs) is still debatable, it is widely accepted that GBM has a small population of cells expressing CSC markers (~1%) that are highly resistant to chemo-radiation therapy. Recent evidence indicates that hypoxia induces cancer stem cell (CSC) phenotypes via epithelial-to-mesenchymal transition (EMT) that promote therapeutic resistance in solid tumours. Given that GBMs are extensively hypooxygenated heterogenous tumours, understanding the molecular relationship between hypoxia, biology of CSCs, EMT and chemoresistance would be invaluable for development of drugs that can target CSCs. Evidence suggests that hypoxia inducible factors (HIFs), NF-B and aldehyde dehydrogenase (ALDH) together orchestrate the stemness and chemoresistance in hypoxia induced CSCs. But the insights on the mechanisms still remain obscure. In this study we used an in vitro GBM CSC and hypoxia model along with NF-B-p65 and HIF transfected GBM cell lines to investigate the relationship between HIFs, NF-B activation and ALDH activity and their role in chemoresistance. The findings of this study demonstrated that GBM cells grown as spheres consist of a vast proportion of hypoxic cells with elevated CSC and EMT markers suggesting hypoxia induced EMT. GBM-CSCs are chemoresistant and displayed increased levels of HIFs, NF-B and ALDH activity. It was also observed that stable transfection of GBM cells with NF-B-p65 or HIFs induced CSC and EMT markers indicating their essential role in maintaining CSC phenotypes. The study also highlighted the importance of NF-B and ALDH in driving chemoresistance and the potential role of NF-B as the master regulator of hypoxia induced stemness in GBM cells. In this study, we used Disulfiram (DS), an anti-alcoholism drug, in combination with copper (Cu) to target the hypoxia-NF-B axis and inhibit ALDH activity to reverse chemoresistance in GBM CSCs. We showed that DS/Cu is cytotoxic to GBM cells and completely eradicated the resistant CSC population at low nanomolar levels in vitro. We also demonstrated that DS/Cu effectively inhibited GBM in vivo using newly formulated PLGA-DS nanoparticles. DS is an FDA approved drug with low/no toxicity to normal tissues and can freely pass through the blood brain barrier (BBB). Further study may lead to quick translation of DS into clinical trials.

Human neurosphere stem cells offer promising potential for the treatment of neurodegenerative diseases. Their well characterized multi-potency of differentiating into neurons, astrocytes, and oligodendrocytes when exposed to the optimum exogenous growth factors make them an exciting area of study (38). Finding novel endogenous methods of modulating stem cell metabolism will allow for the safer treatment of various brain disorders (34). In this experiment, melatonin, N-acetylserotonin, L-tryptophan, and L-DOPA are added in three different concentrations to neurospheres suspended in HNSC/GBM media with less than optimal concentrations of exogenous epidermal growth factor (EGF) and fibroblast growth factor (FGF). The alamarBlue assay (resazurin) was chosen as the most suitable assay for measuring neurosphere metabolism. Metabolic neural stem cells would cause the greatest reduction of the oxidized alamarBlue reagent (resazurin?resorufin), which was detected by a fluorescent plate reader (39-41). The percent reduction in alamarBlue was calculated for all four molecules at three different concentrations and compared to controls without any molecule. Our results illustrate that there was no statistically significant difference at p<0.05 between the biological molecules and the control group except for two exceptions (labeled with asterisks on figures 3 and 5) L-DOPA at a 40 micromolar concentration after 4 hours of incubation and melatonin at a 40 micromolar concentration after 52 hours of incubation.

O glioblastoma multiforme (GBM) é um dos tumores mais letais e a radioterapia permanece como um dos principais tratamentos. Novas estratégias são necessárias para coibir a resistência ao tratamento, como o silenciamento de fatores de transcrição (FTs). Nossa hipótese é a de que FTs associados a listas de genes diferencialmente expressos, os quais foram selecionados para linhagens de GBM irradiadas, ou comparando amostras de GBM à amostras de tecido cerebral, possam fornecer alvos moleculares que aumentariam a morte das células tumorais, quando silenciados. Foram analisadas a proliferação, morte e ciclo celular, além da formação e diferenciação de neuroesferas, utilizando, em quase todas as etapas, a citometria de fluxo. Os FTs HEB e E2F1, cujas funções principais estão relacionadas à neurogênese e proliferação celular, foram selecionados a partir das análises in silico de GBM irradiados ou não, ou de GBMs comparados a amostras de cérebro normal, respectivamente. Esses FTs encontram-se expressos em linhagens U87, astrócitos primários e neuroesferas provenientes das mesmas, analisadas por Western blot. O silenciamento de HEB e E2F1 na linhagem U87, de forma geral, reduziu a proliferação, induziu morte celular e diminuiu a porcentagem de células em G0/ G1, em pelo menos um dos tempos analisados (24, 48 e 72h) em relação ao grupo transfectado com a sequência scrambled. O silenciamento de HEB e E2F1 reduziu o número de neuroesferas quando comparadas às células transfectadas com a sequência scrambled. Possivelmente, a capacidade anti-proliferativa do silenciamento dos FTs HEB e E2F1 observada no cultivo em monocamada da U87, possam atuar na capacidade de formação de neuroesferas e, consequentemente, podem ter um papel na manutenção das células tronco do GBM. O silenciamento não alterou a radiorresistência da U87 cultivada em monocamada, com exceção dos efeitos do silenciamento de E2F1 em 24 h, em que houve radioproteção. A irradiação não reduziu o número de neuroesferas silenciadas para HEB em comparação ao grupo não irradiado, mas reduziu o número de células presentes nas neuroesferas, indicando uma possível atuação de HEB na resposta à irradiação em neuroesferas, fato este nunca antes descrito. O silenciamento de E2F1 não interferiu na resposta das neuroesferas à radiação. A expressão de CD133 avaliada oito dias após a dissociação das células silenciadas para E2F1 e HEB, cultivadas em meio de diferenciação, foram superiores ao do grupo scrambled, indicando uma possível diminuição na diferenciação celular. O silenciamento dos dois FTs não atuou na seleção positiva de CD133+ após a irradiação, como observado no grupo das neuroesferas transfectadas com a sequência scrambled e irradiadas, comparado às não irradiadas. Assim, E2F1 e HEB mostraram-se alvos interessantes no sentido de reduzir a proliferação, tanto em células U87 cultivadas em monocamada quanto em neuroesferas. / Glioblastoma multiforme (GBM) is one of the most lethal tumors, and radiation therapy remains one of the main treatments. New strategies are needed to suppress typical GBM treatment resistance and transcription factors (TFs) silencing seems to be a promising strategy. Our hypothesis is that TFs associated with lists of differentially expressed genes which were selected for irradiated compared to shamirradiated GBM cell lines, or GBM samples compared to brain tissue samples, could provide molecular targets that are supposed to increase tumor cell death when they are silenced. We analyzed proliferation, cell death and cell cycle progression, besides the formation and differentiation of neurospheres, using several analyses by flow cytometry. The TFs HEB and E2F1, whose primary functions are related to neurogenesis and cell proliferation, were selected from in silico analysis of GBM irradiated or sham-irradiated GBMs and GBM samples compared with normal brain samples, respectively. These TFs were found expressed in U87 GBM cell line, and primary astrocytes, as well as in neurospheres derivated from both, as analyzed by Western blot. Silencing of E2F1 and HEB in U87 cells, reduced proliferation, induced cell death and decreased the percentage of cells at G0/G1 (24, 48 or 72h) compared to the scrambled sequence transfected group. HEB and E2F1 silencing reduced the number of neurospheres when compared to cells transfected with scrambled sequence. Possibly, the anti-proliferative ability of silencing of HEB and E2F1 TFs observed in monolayer culture of U87, may act in neurospheres forming capacity and therefore may play a role in the maintenance of GBM stem cells. In our experiments, gene silencing did not alter the radio-resistance of U87 grown in monolayer. Irradiation did not reduce the number of neurospheres silenced for HEB compared to non-irradiated group, but reduced the number of cells present in neurospheres, indicating a possible role of HEB in response to ionizing irradiation in neurospheres, a fact that was not described yet. The silencing of E2F1 in neurospheres did not affect the response to irradiation. The expression of CD133, as assessed at eight days after the dissociation of cells silenced for E2F1 and HEB (cultured in differentiation culture media), was superior compared with the scrambled group, indicating a possible decrease in cell differentiation. The silencing of both TFs did not influence the positive selection of CD133 after irradiation, as observed in the group of neurospheres transfected with scrambled sequence, and irradiated compared to nonirradiated. Thus, E2F1 and HEB proved to be interesting targets for decreasing proliferation in both U87 cells grown as monolayer or neurospheres.

O glioblastoma multiforme (GBM) é um dos tumores mais letais e a radioterapia permanece como um dos principais tratamentos. Novas estratégias são necessárias para coibir a resistência ao tratamento, como o silenciamento de fatores de transcrição (FTs). Nossa hipótese é a de que FTs associados a listas de genes diferencialmente expressos, os quais foram selecionados para linhagens de GBM irradiadas, ou comparando amostras de GBM à amostras de tecido cerebral, possam fornecer alvos moleculares que aumentariam a morte das células tumorais, quando silenciados. Foram analisadas a proliferação, morte e ciclo celular, além da formação e diferenciação de neuroesferas, utilizando, em quase todas as etapas, a citometria de fluxo. Os FTs HEB e E2F1, cujas funções principais estão relacionadas à neurogênese e proliferação celular, foram selecionados a partir das análises in silico de GBM irradiados ou não, ou de GBMs comparados a amostras de cérebro normal, respectivamente. Esses FTs encontram-se expressos em linhagens U87, astrócitos primários e neuroesferas provenientes das mesmas, analisadas por Western blot. O silenciamento de HEB e E2F1 na linhagem U87, de forma geral, reduziu a proliferação, induziu morte celular e diminuiu a porcentagem de células em G0/ G1, em pelo menos um dos tempos analisados (24, 48 e 72h) em relação ao grupo transfectado com a sequência scrambled. O silenciamento de HEB e E2F1 reduziu o número de neuroesferas quando comparadas às células transfectadas com a sequência scrambled. Possivelmente, a capacidade anti-proliferativa do silenciamento dos FTs HEB e E2F1 observada no cultivo em monocamada da U87, possam atuar na capacidade de formação de neuroesferas e, consequentemente, podem ter um papel na manutenção das células tronco do GBM. O silenciamento não alterou a radiorresistência da U87 cultivada em monocamada, com exceção dos efeitos do silenciamento de E2F1 em 24 h, em que houve radioproteção. A irradiação não reduziu o número de neuroesferas silenciadas para HEB em comparação ao grupo não irradiado, mas reduziu o número de células presentes nas neuroesferas, indicando uma possível atuação de HEB na resposta à irradiação em neuroesferas, fato este nunca antes descrito. O silenciamento de E2F1 não interferiu na resposta das neuroesferas à radiação. A expressão de CD133 avaliada oito dias após a dissociação das células silenciadas para E2F1 e HEB, cultivadas em meio de diferenciação, foram superiores ao do grupo scrambled, indicando uma possível diminuição na diferenciação celular. O silenciamento dos dois FTs não atuou na seleção positiva de CD133+ após a irradiação, como observado no grupo das neuroesferas transfectadas com a sequência scrambled e irradiadas, comparado às não irradiadas. Assim, E2F1 e HEB mostraram-se alvos interessantes no sentido de reduzir a proliferação, tanto em células U87 cultivadas em monocamada quanto em neuroesferas. / Glioblastoma multiforme (GBM) is one of the most lethal tumors, and radiation therapy remains one of the main treatments. New strategies are needed to suppress typical GBM treatment resistance and transcription factors (TFs) silencing seems to be a promising strategy. Our hypothesis is that TFs associated with lists of differentially expressed genes which were selected for irradiated compared to shamirradiated GBM cell lines, or GBM samples compared to brain tissue samples, could provide molecular targets that are supposed to increase tumor cell death when they are silenced. We analyzed proliferation, cell death and cell cycle progression, besides the formation and differentiation of neurospheres, using several analyses by flow cytometry. The TFs HEB and E2F1, whose primary functions are related to neurogenesis and cell proliferation, were selected from in silico analysis of GBM irradiated or sham-irradiated GBMs and GBM samples compared with normal brain samples, respectively. These TFs were found expressed in U87 GBM cell line, and primary astrocytes, as well as in neurospheres derivated from both, as analyzed by Western blot. Silencing of E2F1 and HEB in U87 cells, reduced proliferation, induced cell death and decreased the percentage of cells at G0/G1 (24, 48 or 72h) compared to the scrambled sequence transfected group. HEB and E2F1 silencing reduced the number of neurospheres when compared to cells transfected with scrambled sequence. Possibly, the anti-proliferative ability of silencing of HEB and E2F1 TFs observed in monolayer culture of U87, may act in neurospheres forming capacity and therefore may play a role in the maintenance of GBM stem cells. In our experiments, gene silencing did not alter the radio-resistance of U87 grown in monolayer. Irradiation did not reduce the number of neurospheres silenced for HEB compared to non-irradiated group, but reduced the number of cells present in neurospheres, indicating a possible role of HEB in response to ionizing irradiation in neurospheres, a fact that was not described yet. The silencing of E2F1 in neurospheres did not affect the response to irradiation. The expression of CD133, as assessed at eight days after the dissociation of cells silenced for E2F1 and HEB (cultured in differentiation culture media), was superior compared with the scrambled group, indicating a possible decrease in cell differentiation. The silencing of both TFs did not influence the positive selection of CD133 after irradiation, as observed in the group of neurospheres transfected with scrambled sequence, and irradiated compared to nonirradiated. Thus, E2F1 and HEB proved to be interesting targets for decreasing proliferation in both U87 cells grown as monolayer or neurospheres.

在成年的哺乳類動物大腦中有兩個區域，可以不斷的有新的神經細胞生成，一個位於大腦側腦室旁內側(Subventricular zone of anterior lateral ventricle ;SVZ)，另一個位於海馬迴(hippocampus)內的齒狀迴(Subgran- ular zone of dentate gyrus ;SGZ) ，其中海馬迴是本論文主要探討的腦區。 神經前驅細胞(Neural progenitor cells :NPC)因具有自我更新(self -renewal）、增殖(proliferative）、多能(multipotent）的能力以及遷移性(Migration)，所以可利用海馬迴內生性的神經前驅細胞(NPC)，促進其增殖以替代因損傷、老化或疾病而損失的神經細胞。神經前驅細胞經由細胞體外培養過程會形成神經球(Neurospheres)，神經球和神經前驅細胞同樣具有自我更新以及可以分化成其他神經細胞的能力。 本研究觀察到，對成年神經新生進行體外藥物的篩選中，化合物Chemical-X，有明顯的促進神經新生的能力。實驗中取健康成年雄性大鼠為實驗動物，分離出成年大鼠之海馬迴神經前驅細胞。用Chemical-X處理後，觀察神經球自我更新能力，以及再把新生成的神經球利用免疫螢光染色處理，瞭解神經前驅細胞經藥物處理後所新生成的細胞，是否仍維持在神經前驅細胞的狀態。進而評估藥物能否達到促進神經新生的目的。