A Doença de Alzheimer (DA) é uma desordem neurodegenerativa caracterizada por um declínio cognitivo global, incluindo uma perda progressiva de memória, orientação e raciocínio, associada com a degeneração especifica de neurônios colinérgicos. As alterações histopatológicas que definem a DA são protéicos de β-amilóide e tau hiperfosforilada em placas senis e emaranhados neurofibrilares, respectivamente. Porém, os mecanismos moleculares que levam a DA não estão bem caracterizados e ainda não há nenhum tratamento eficaz para prevenir ou reverter os seus sintomas. Parte dessa dificuldade se deve à escassez de modelos experimentais adequados. Previamente nosso grupo estabeleceu condições experimentais para a diferenciação do neuroblastoma humano SH-SY5Y em neurônios dopaminérgicos a partir da adição de ácido retinóico (AR). Alguns estudos sugerem que esta linhagem, quando tratada com AR e fator neurotrófico derivado do cérebro (BDNF), apresenta uma mudança para um fenótipo colinérgico. Dessa forma, este trabalho tem por objetivo caracterizar a diferenciação colinérgica desta linhagem e estabelecer as melhores condições de tratamento com ácido ocadáico (AO), que promove a hiperfosforilação e deposição da proteína tau, em combinação com oligômeros solúveis de β-amilóide (Aβ1-42), para uso como modelo in vitro ao estudo da DA. As células proliferativas foram cultivadas em meio DMEM/F12 com 10% de soro fetal ( FB). A diferenciação foi induzida com 10μM de AR em meio a cultura com 1% de SFB durante sete dias, sendo o BDNF (10 nM) acrescentado a partir do quarto dia. Determinamos a atividade da Acetilcolinesterase (AChE), da Colina Acetiltransferase (ChAT) e o imunoconteúdo de ChAT como marcadores colinérgicos. O co-tratamento com BDNF resultou em células com notável morfologia neuronal, apresentando aumento na densidade e comprimento de neuritos e nos marcadores colinérgicos. Após o estabelecimento do modelo colinérgico, as células diferenciadas da linhagem de neuroblastoma SH-SY5Y foram desafiadas com uma curva de dose das neurotoxinas Aβ1-42 ou AO, e a neurotoxicidade foi avaliada pelo ensaio do brometo de 3-(4,5-dimetiltiazol-2il)-2,5-difeniltetrazolium (MTT) em combinação com a densidade de neuritos. A partir desses resultados, doses subletais das toxinas foram selecionadas para estabelecer o modelo in vitro da Doença de Alzheimer. Esses resultados fazem dessa linhagem celular uma ferramenta útil no campo da neurociência, podendo torná-la um modelo adequado para o estudo da Doença de Alzheimer. / Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a global cognitive decline, including progressive loss of memory, orientation and reasoning associated with a specific degeneration of cholinergic neurons. Histopathological changes that define AD are amyloid-β hyperphosphorylated tau deposits in senile plaques and neurofibrillary tangles, respectively. However, the leading molecular mechanisms are not well elucidated and still there is no effective treatment to prevent or reverse the symptoms. Part of this difficulty is due to the lack of suitable experimental models. Our group previously established experimental conditions for the differentiation of human neuroblastoma SH-SY5Y cells into dopaminergic neurons-like with retinoic acid (RA) addition. Some studies suggested that when treated with RA and brain-derived neurotrophic factor (BDNF) this cell line has a shift to a cholinergic like phenotype. Thus, this study aims to characterize the cholinergic differentiation of SH-SY5Y cell line and to determine the best conditions for okadaic acid (OA) treatment, which promotes the deposition and hyperphosphorylation of tau protein, in combination with solubleoligomers of β- myl (Aβ - 42). Thereby we established a suitable AD in vitro model. Exponentially growing SH-SY5Y cells were maintained in DMEM / F12 medium with 10% fetal bovine serum (FBS). Differentiation was triggered by the combination of 10 μM of RA plus medium with 1% of FBS during 7 days, BDNF was added on 4th day. We determined Acetylcholinesterase (AChE) and Choline Acetyltransferase (ChAT) enzymatic activities and the ChAT immunocontent as cholinergic markers. Once the cholinergic like in vitro model was established, the differentiated SH- Y5Y cells were challeng with of Aβ -42 or AO dose curve, and neurotoxicity was evaluated by an 3-(4,5-dimethylthiazol-2il)-2,5-diphenyltetrazolium bromide (MTT) assay in combination with neurite density. Hence, sublethal doses of neurotoxins were selected to determine the in vitro model of Alzheimer's disease. Taken together, these results suggested SH-SY5Y cell line as a useful and suitable model in Alzheimer's disease research.

Cancer is one of the most challenging problems the modern medicine is facing today. An increasing incidence and a great variability of tumor cells are the main reasons those drive the research to develop better diagnostics and therapeutic protocols. Histone deacetylase inhibitors, a group of epigenetic chemotherapeutics, are able to improve the performance of currently used anticancer agents. Vaplroic acid that is commonly used as antiepileptic drug exhibits a remarkable anticancer activity by itself as well as it is capable of therapy potentiation based on other therapeutic agents. Its effect to inhibit growth of tumor cells and induce apoptotic cell death seems to be even greater under hypoxic conditions (<1% O2). This study is focused on effect of valproic acid on neuroblastoma cell lines in vitro under normoxic and hypoxic conditions. We observed significantly greater efficacy of valproic acid in hypoxia compared to normoxia. The mechanism of induction of apoptotic cell death is based on disruption of the balance between pro- and antiapoptoic proteins. Intrinsic apoptotic pathway is probably initiated by the action of 19 kDa variant of proapoptotic protein Bax on mitochondrial membrane. Moreover, we examined the efficiency of a combined treatment of neuroblastoma cells with valproic acid and...

A Doença de Alzheimer (DA) é uma desordem neurodegenerativa caracterizada por um declínio cognitivo global, incluindo uma perda progressiva de memória, orientação e raciocínio, associada com a degeneração especifica de neurônios colinérgicos. As alterações histopatológicas que definem a DA são protéicos de β-amilóide e tau hiperfosforilada em placas senis e emaranhados neurofibrilares, respectivamente. Porém, os mecanismos moleculares que levam a DA não estão bem caracterizados e ainda não há nenhum tratamento eficaz para prevenir ou reverter os seus sintomas. Parte dessa dificuldade se deve à escassez de modelos experimentais adequados. Previamente nosso grupo estabeleceu condições experimentais para a diferenciação do neuroblastoma humano SH-SY5Y em neurônios dopaminérgicos a partir da adição de ácido retinóico (AR). Alguns estudos sugerem que esta linhagem, quando tratada com AR e fator neurotrófico derivado do cérebro (BDNF), apresenta uma mudança para um fenótipo colinérgico. Dessa forma, este trabalho tem por objetivo caracterizar a diferenciação colinérgica desta linhagem e estabelecer as melhores condições de tratamento com ácido ocadáico (AO), que promove a hiperfosforilação e deposição da proteína tau, em combinação com oligômeros solúveis de β-amilóide (Aβ1-42), para uso como modelo in vitro ao estudo da DA. As células proliferativas foram cultivadas em meio DMEM/F12 com 10% de soro fetal ( FB). A diferenciação foi induzida com 10μM de AR em meio a cultura com 1% de SFB durante sete dias, sendo o BDNF (10 nM) acrescentado a partir do quarto dia. Determinamos a atividade da Acetilcolinesterase (AChE), da Colina Acetiltransferase (ChAT) e o imunoconteúdo de ChAT como marcadores colinérgicos. O co-tratamento com BDNF resultou em células com notável morfologia neuronal, apresentando aumento na densidade e comprimento de neuritos e nos marcadores colinérgicos. Após o estabelecimento do modelo colinérgico, as células diferenciadas da linhagem de neuroblastoma SH-SY5Y foram desafiadas com uma curva de dose das neurotoxinas Aβ1-42 ou AO, e a neurotoxicidade foi avaliada pelo ensaio do brometo de 3-(4,5-dimetiltiazol-2il)-2,5-difeniltetrazolium (MTT) em combinação com a densidade de neuritos. A partir desses resultados, doses subletais das toxinas foram selecionadas para estabelecer o modelo in vitro da Doença de Alzheimer. Esses resultados fazem dessa linhagem celular uma ferramenta útil no campo da neurociência, podendo torná-la um modelo adequado para o estudo da Doença de Alzheimer. / Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a global cognitive decline, including progressive loss of memory, orientation and reasoning associated with a specific degeneration of cholinergic neurons. Histopathological changes that define AD are amyloid-β hyperphosphorylated tau deposits in senile plaques and neurofibrillary tangles, respectively. However, the leading molecular mechanisms are not well elucidated and still there is no effective treatment to prevent or reverse the symptoms. Part of this difficulty is due to the lack of suitable experimental models. Our group previously established experimental conditions for the differentiation of human neuroblastoma SH-SY5Y cells into dopaminergic neurons-like with retinoic acid (RA) addition. Some studies suggested that when treated with RA and brain-derived neurotrophic factor (BDNF) this cell line has a shift to a cholinergic like phenotype. Thus, this study aims to characterize the cholinergic differentiation of SH-SY5Y cell line and to determine the best conditions for okadaic acid (OA) treatment, which promotes the deposition and hyperphosphorylation of tau protein, in combination with solubleoligomers of β- myl (Aβ - 42). Thereby we established a suitable AD in vitro model. Exponentially growing SH-SY5Y cells were maintained in DMEM / F12 medium with 10% fetal bovine serum (FBS). Differentiation was triggered by the combination of 10 μM of RA plus medium with 1% of FBS during 7 days, BDNF was added on 4th day. We determined Acetylcholinesterase (AChE) and Choline Acetyltransferase (ChAT) enzymatic activities and the ChAT immunocontent as cholinergic markers. Once the cholinergic like in vitro model was established, the differentiated SH- Y5Y cells were challeng with of Aβ -42 or AO dose curve, and neurotoxicity was evaluated by an 3-(4,5-dimethylthiazol-2il)-2,5-diphenyltetrazolium bromide (MTT) assay in combination with neurite density. Hence, sublethal doses of neurotoxins were selected to determine the in vitro model of Alzheimer's disease. Taken together, these results suggested SH-SY5Y cell line as a useful and suitable model in Alzheimer's disease research.

A Doença de Alzheimer (DA) é uma desordem neurodegenerativa caracterizada por um declínio cognitivo global, incluindo uma perda progressiva de memória, orientação e raciocínio, associada com a degeneração especifica de neurônios colinérgicos. As alterações histopatológicas que definem a DA são protéicos de β-amilóide e tau hiperfosforilada em placas senis e emaranhados neurofibrilares, respectivamente. Porém, os mecanismos moleculares que levam a DA não estão bem caracterizados e ainda não há nenhum tratamento eficaz para prevenir ou reverter os seus sintomas. Parte dessa dificuldade se deve à escassez de modelos experimentais adequados. Previamente nosso grupo estabeleceu condições experimentais para a diferenciação do neuroblastoma humano SH-SY5Y em neurônios dopaminérgicos a partir da adição de ácido retinóico (AR). Alguns estudos sugerem que esta linhagem, quando tratada com AR e fator neurotrófico derivado do cérebro (BDNF), apresenta uma mudança para um fenótipo colinérgico. Dessa forma, este trabalho tem por objetivo caracterizar a diferenciação colinérgica desta linhagem e estabelecer as melhores condições de tratamento com ácido ocadáico (AO), que promove a hiperfosforilação e deposição da proteína tau, em combinação com oligômeros solúveis de β-amilóide (Aβ1-42), para uso como modelo in vitro ao estudo da DA. As células proliferativas foram cultivadas em meio DMEM/F12 com 10% de soro fetal ( FB). A diferenciação foi induzida com 10μM de AR em meio a cultura com 1% de SFB durante sete dias, sendo o BDNF (10 nM) acrescentado a partir do quarto dia. Determinamos a atividade da Acetilcolinesterase (AChE), da Colina Acetiltransferase (ChAT) e o imunoconteúdo de ChAT como marcadores colinérgicos. O co-tratamento com BDNF resultou em células com notável morfologia neuronal, apresentando aumento na densidade e comprimento de neuritos e nos marcadores colinérgicos. Após o estabelecimento do modelo colinérgico, as células diferenciadas da linhagem de neuroblastoma SH-SY5Y foram desafiadas com uma curva de dose das neurotoxinas Aβ1-42 ou AO, e a neurotoxicidade foi avaliada pelo ensaio do brometo de 3-(4,5-dimetiltiazol-2il)-2,5-difeniltetrazolium (MTT) em combinação com a densidade de neuritos. A partir desses resultados, doses subletais das toxinas foram selecionadas para estabelecer o modelo in vitro da Doença de Alzheimer. Esses resultados fazem dessa linhagem celular uma ferramenta útil no campo da neurociência, podendo torná-la um modelo adequado para o estudo da Doença de Alzheimer. / Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a global cognitive decline, including progressive loss of memory, orientation and reasoning associated with a specific degeneration of cholinergic neurons. Histopathological changes that define AD are amyloid-β hyperphosphorylated tau deposits in senile plaques and neurofibrillary tangles, respectively. However, the leading molecular mechanisms are not well elucidated and still there is no effective treatment to prevent or reverse the symptoms. Part of this difficulty is due to the lack of suitable experimental models. Our group previously established experimental conditions for the differentiation of human neuroblastoma SH-SY5Y cells into dopaminergic neurons-like with retinoic acid (RA) addition. Some studies suggested that when treated with RA and brain-derived neurotrophic factor (BDNF) this cell line has a shift to a cholinergic like phenotype. Thus, this study aims to characterize the cholinergic differentiation of SH-SY5Y cell line and to determine the best conditions for okadaic acid (OA) treatment, which promotes the deposition and hyperphosphorylation of tau protein, in combination with solubleoligomers of β- myl (Aβ - 42). Thereby we established a suitable AD in vitro model. Exponentially growing SH-SY5Y cells were maintained in DMEM / F12 medium with 10% fetal bovine serum (FBS). Differentiation was triggered by the combination of 10 μM of RA plus medium with 1% of FBS during 7 days, BDNF was added on 4th day. We determined Acetylcholinesterase (AChE) and Choline Acetyltransferase (ChAT) enzymatic activities and the ChAT immunocontent as cholinergic markers. Once the cholinergic like in vitro model was established, the differentiated SH- Y5Y cells were challeng with of Aβ -42 or AO dose curve, and neurotoxicity was evaluated by an 3-(4,5-dimethylthiazol-2il)-2,5-diphenyltetrazolium bromide (MTT) assay in combination with neurite density. Hence, sublethal doses of neurotoxins were selected to determine the in vitro model of Alzheimer's disease. Taken together, these results suggested SH-SY5Y cell line as a useful and suitable model in Alzheimer's disease research.

Neuroblastoma, a neuroendocrine tumor, is the most common cancer in infancy. 75 % of those affected are under the age of 5. The disease is heterogeneous and survival rate is low.   Current treatment of neuroblastoma consists of surgery, radiation and chemotherapy, where the targets for the treatment are the malign cells. Due to the cancer cells instable genome there is a risk for resistance development. This negatively impacts the treatments goal of hindering tumor growth and spread.  Tumor growth is not only determined by malign cells but also the interactions of those tumor cells with tumor vessels and different types of cells in the tumor stroma.   The aim of this paper is to develop a relevant histological method to study the properties of tumor stroma in tumor sections retrieved from human NB tumor xenografts in mice treated with angiogenesis inhibitors SU11657 and Zoledronic acid. The study is a continuation of previous studies with the inhibitors which have shown good effect on tumor growth and angiogenesis on neuroblastoma.   In the short term treatment with SU11657 and Zoledron acid showed that tumor growth declined. In the longer treatment with SU11657 the growth didn’t decline with the same rate compared to the short term treatment. Angiogenesis on the other hand decreased in all the treatments independent of treatment duration. The histological staining with Sirius red revealed that treated tumors had an increased amount of stroma compared to the untreated tumors.   In conclusion the relative increase of tumor volume, decreased number of vessels and expansion of tumor stroma in the longer treatment with SU11657 indicated that tumors might survive the angiogenesis inhibitor treatment through expansion/activation of its stroma. The histological staining with Sirius red in saturated picric acid marked the collagen, i.e. stroma, well and enabled quantification of the stroma.

[EN] In neuroblastoma management various medical imaging modalities are involved. Among them, scans of 123I-mBG continue being today the most specific and sensitive, so they have an important role in assessing the stage of the disease and the patient's response to treatment. Although guidelines that attempt to homogenize the process of patient preparation and image acquisition are followed when performing the scan, there are intrinsic and hardly avoidable problems to this type of medical imaging that degrade its quality. Images acquired, especially in young children, show poor defined contours, low contrast and intensity variations between scans, making it difficult to identify the areas of uptake of 123I-mIBG. In the recent years, various semi quantitative scoring methods of 123ImIBG scans have emerged in order to quantify the effectiveness of treatment response and to standardize the assessment of the studies. These methods have proven to be able to assess patient prognosis, so that they have become essential when assessing the disease. But when applying any scoring method, and because the poor definition of the image, the valuation may still be too subjective and dependent on the observer, making difficult the comparison of results between different medical institutions. It is therefore an automated tool required to assist the diagnosis which minimizes subjectivity in the application of these scoring methods and improves the objective evaluation of the response to treatment and the prognosis of the patient. In this work we propose a methodology to take objective and easily comparable measurements on 123I-mIBG scans, along with an image processing algorithm to automatically detect 123I-mIBG uptake areas which are sufficiently anomalous to be suspected of being pathological. / [ES] En el tratamiento del neuroblastoma intervienen varias modalidades de imagen médica. Entre ellas, las gammagrafías de 123I-mIBG siguen destacando en la actualidad por ser las más específicas y sensibles, por lo que tienen un papel importante en la evaluación del estadio de la enfermedad y la respuesta del paciente al tratamiento. Aunque a la hora de realizar la gammagrafía se siguen las directrices que intentan homogeneizar el proceso de preparación del paciente y de adquisición de la imagen, existen problemas intrínsecos a esta modalidad de imagen médica que degradan su calidad y que son difícilmente evitables. Las imágenes adquiridas, especialmente en niños pequeños, presentan pobre resolución, contornos mal definidos, bajo contraste y variaciones en la intensidad entre pruebas sucesivas, lo que dificulta la identificación de los focos de captación de 123I-mIBG. En los últimos años, han surgido diversos métodos semicuantitativos de puntuación de las gammagrafías de 123I-mIBG con el fin de cuantificar la eficacia de la respuesta al tratamiento y uniformizar la evaluación de los estudios. Estos métodos han demostrado tener poder para evaluar el pronóstico del paciente, por lo que se han convertido en imprescindibles a la hora de evaluar la enfermedad. Pero a la hora de aplicar cualquier método de puntuación, y debido a la pobre definición de la imagen, la valoración aún puede ser demasiado subjetiva y dependiente del observador, dificultando la comparación de resultados entre diferentes instituciones médicas. Es por tanto que es necesaria una herramienta automática de ayuda al diagnóstico que reduzca la subjetividad a la hora de aplicar estos métodos de puntuación y así mejorar la evaluación objetiva de la respuesta al tratamiento y el pronóstico del paciente. En este trabajo proponemos una metodología para tomar mediciones objetivas y fácilmente comparables sobre las gammagrafías de 123I-mIBG, junto con un algoritmo de procesamiento de la imagen para detectar de forma automática las zonas de captación de 123I-mIBG que son suficientemente anómalas para ser sospechosas de ser patológicas. / [CAT] En el tractament del neuroblastoma intervenen diverses modalitats d'imatge mèdica. Entre elles, les gammagrafies de 123I-mIBG segueixen destacant en l'actualitat per ser les més específiques i sensibles, per la qual cosa tenen un paper important en l'avaluació de l'estadi de la malaltia i la resposta del pacient al tractament. Encara que a l'hora de realitzar la gammagrafia se segueixen les directrius que intenten homogeneïtzar el procés de preparació del pacient i d'adquisició de la imatge, existeixen problemes intrínsecs a aquesta modalitat d'imatge mèdica que degraden la seua qualitat i que són difícilment evitables. Les imatges adquirides, especialment en xiquets xicotets, presenten pobra resolució, contorns mal definits, sota contrast i variacions en la intensitat entre proves successives, la qual cosa dificulta la identificació dels focus de captació de 123I-mIBG. En els últims anys, han sorgit diversos mètodes semicuantitativos de puntuació de les gammagrafies de 123I-mIBG amb la finalitat de quantificar l'eficàcia de la resposta al tractament i uniformitzar l'avaluació dels estudis. Aquests mètodes han demostrat tenir poder per a avaluar el pronòstic del pacient, per la qual cosa s'han convertit en imprescindibles a l'hora d'avaluar la malaltia. Però a l'hora d'aplicar qualsevol mètode de puntuació, i a causa de la pobra definició de la imatge, la valoració encara pot ser massa subjectiva i dependent de l'observador, dificultant la comparació de resultats entre diferents institucions mèdiques. És per tant que és necessària una eina automàtica d'ajuda al diagnòstic que reduïsca la subjectivitat a l'hora d'aplicar aquests mètodes de puntuació i així millorar l'avaluació objectiva de la resposta al tractament i el pronòstic del pacient. En aquest treball proposem una metodologia per a prendre mesuraments objectius i fàcilment comparables sobre les gammagrafies de 123I-mIBG, juntament amb un algorisme de processament de la imatge per a detectar de forma automàtica les zones de captació de 123I-mIBG que són suficientment anòmales per a ser sospitoses de ser patològiques. / Martínez Díaz, R. (2015). Análisis, propuesta y desarrollo de técnicas de medición objetiva en imágenes 123/-mIBG, para pacientes con neuroblastoma [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/58984 / TESIS

Le neuroblastome est le cancer solide extra-cranial le plus fréquent chez l’enfant. Il est caractérisé par une très grande hétérogénéité tant au niveau clinique que moléculaire. Alors que certains patients rentrent spontanément en rémission, on peut se demander quels facteurs permettent la réémergence du cancer chez d’autres malgré traitement. Pour répondre à cette question, il convient d’identifier chez les patients ayant rechuté, les différentes populations clonales coexistant au diagnostic et/ou à la rechute. Cela permet, entre autre, d’étudier les voies différemment altérées entre ces deux temps. Dans cette optique, nous présentons ici QuantumClone, un algorithme de reconstruction clonal à partir de données de séquençage, ainsi que son application à une cohorte de patients souffrant d’un neuroblastome. Sur ces données, l’application de notre méthode a permis d’identifier des différences dans le ratio de variants prédits fonctionnels par rapport à ceux prédits passagers entre les populations ancestrales, enrichies à la rechute ou appauvries à la rechute. / Neuroblastoma is the most frequent solid extra-cranial cancer of childhood. This cancer displays a high heterogeneity both at clinical and molecular levels. Even though in some patients spontaneous remission can be observed, some others relapse despite treatment and surgical resection. It may be wondered which are the factors that distinguish these two cases. In order to answer this question, identification of populations coexisting at diagnosis and/or relapse in the patients which have relapsed is a prerequisite. This would allow, between other things, to study the pathways differently altered in clones that are specific to each time point. With this in mind, we hereby present QuantumClone, a clonal reconstruction algorithm from sequencing data. In addition, we applied this method to a cohort of patients suffering from neuroblastoma. On these data, our method identified differences in the functional mutation rate, i.e. the number of putative functional variants by total number of variants, between the ancestral clones, clones expanding at relapse, and clones shrinking at relapse.

Neuroblastoma (NB) is the most common extracranial solid tumour in childhood. NB is thought to arise from the failed differentiation of neural crest progenitor cells that would normally form tissues of the adrenal gland and sympathetic nervous system. These neural crest progenitors then uncontrollably proliferate forming a tumour. Despite aggressive surgery and chemotherapy, the cure rate of high-risk NB patients remains below 30%. Our laboratory has shown that human NB tumour specimens and high-risk patient derived cell lines express pannexin 1 (PANX1), and that treatment with the PANX1 channel blockers carbenoxolone or probenecid constitute reduce NB progression in vitro and in vivo. PANX1 is a glycoprotein that forms single membrane channels best known to serve as conduits for ATP release. Interestingly, while PANX1 was also detected in control neurons by western blotting, its banding pattern was strikingly different as a band at around 50 kDa was found in all NB cell lines, but not in neurons. Using shRNA targeting PANX1 and deglycosylation enzymes, I have shown that this band corresponds to a PANX1 glycosylated species. PANX1 has been reported to be phosphorylated in NB at amino acid Y10. PANX1 is also predicted to be glycosylated at N255. In order to study the role of these post-translational modifications, myc-tagged Y10F and N255A PANX1 mutants were engineered by site-directed mutagenesis. Immunolocalization and cell surface biotinylation assays suggest that the localization both mutants at the cell surface is reduced compared to that of myc-PANX1. Dye uptake assays revealed that myc-Y10F has significantly reduced channel activity. Expression of myc-Y10F and myc-N255A in NB cells inhibited cell proliferation and decreased metastatic potential in vitro. Further analysis of NB tumour specimens revealed that there is a missense mutation in PANX1 resulting in the formation of truncated peptide (amino acid 1-99). Interestingly, I have found that when co-expressed with myc-PANX1, PANX11-99, reduced PANX1 channel activity. Taken together, these findings indicate that phosphorylation on Y10 and glycosylation on N255 regulate PANX1 channel activity and exacerbate NB malignancy, while the expression of PANX11-99 in NB may be beneficial.

Cancer is one of the most challenging problems the modern medicine is facing today. An increasing incidence and a great variability of tumor cells are the main reasons those drive the research to develop better diagnostics and therapeutic protocols. Histone deacetylase inhibitors, a group of epigenetic chemotherapeutics, are able to improve the performance of currently used anticancer agents. Vaplroic acid that is commonly used as antiepileptic drug exhibits a remarkable anticancer activity by itself as well as it is capable of therapy potentiation based on other therapeutic agents. Its effect to inhibit growth of tumor cells and induce apoptotic cell death seems to be even greater under hypoxic conditions (<1% O2). This study is focused on effect of valproic acid on neuroblastoma cell lines in vitro under normoxic and hypoxic conditions. We observed significantly greater efficacy of valproic acid in hypoxia compared to normoxia. The mechanism of induction of apoptotic cell death is based on disruption of the balance between pro- and antiapoptoic proteins. Intrinsic apoptotic pathway is probably initiated by the action of 19 kDa variant of proapoptotic protein Bax on mitochondrial membrane. Moreover, we examined the efficiency of a combined treatment of neuroblastoma cells with valproic acid and...

Doctor Scientiae / Neuroblastoma is the most common extracranial solid tumor in childhood. The survival rates of patients with neuroblastoma, especially those in the high-risk category, are still low despite varied therapies. The detailed understanding of the molecular mechanisms underlying the pathogenesis of neuroblastoma is essential to develop better therapeutics and improve the poor survival rates. This study provides a multi-omic analysis of neuroblastoma datasets from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) neuroblastoma project and the Gene Expression Omnibus (GEO) data portals to better understand the molecular mechanisms of neuroblastoma.