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O desenvolvimento e a otimização de terapias visando a reposição da função neuronal dependem largamente da melhor compreensão dos mecanismos moleculares por trás da diferenciação de células neurogênicas em neurônios maduros. O ácido retinoico (AR) promove diferenciação neuronal de diversos tipos celulares por meio de reprogramação gênica e de vias de sinalização citoplasmáticas. Os receptores nucleares RXRs são mediadores moleculares fundamentais para os efeitos celulares do AR; no entanto, o papel isolado de cada uma de suas três isoformas na diferenciação neuronal permanece pouco claro. Tendo isso em mente, foi investigada a diferenciação induzida pelo AR na linhagem de neuroblastoma humano SH-SY5Y. A caracterização dos parâmetros induzidos pelo AR nessas células demonstrou parada no ciclo celular e adoção de características típicas de neurônios maduros. Os perfis de expressão das isoformas presentes nas células SH-SY5Y, RXRα e RXRβ, mostraram-se variavelmente regulados ao longo da diferenciação neuronal, tanto a nível de transcritos quanto de proteína. Por fim, o silenciamento transitório e isolado de RXRα e RXRβ durante as primeiras etapas da diferenciação com AR afetou de forma distinta os fenômenos celulares induzidos pelo composto: enquanto RXRα mostra-se fundamental para efeitos genômicos e não genômicos na diferenciação, RXRβ modula negativamente a extensão de neuritos nas células SH-SY5Y. Assim, o trabalho indica a presença de funções distintas para as isoformas de RXR durante as primeiras etapas da diferenciação neuronal induzida pelo AR em neuroblastoma, e traz novas perspectivas para o estudo de RXRs como alvos moleculares nas abordagens clínicas de reposição neuronal. / Developing and optimizing therapies aiming at restoring neuronal function depend on better understanding the molecular mechanisms behind differentiation of neurogenic cells into mature neurons. Retinoic acid (RA) promotes neuronal differentiation in multiple cell types through gene reprogramming and cytosolic signaling pathways. The nuclear RXR receptors are main molecular mediators of RA cellular effects. However, little is known about specific roles of distinct RXR isoforms in neuronal differentiation. In view of this, the RA-mediated differentiation of SH-SY5Y neuroblastoma cell line was investigated. Analysis of RA-induced parameters in SH-SY5Y cells showed cell cycle arrest and adoption of neuronal hallmarks. The expression profiles of RXR isoforms detected in SH-SY5Y cells, RXRα e RXRβ, were found varyingly modulated along neuronal differentiation both at transcript and protein levels. Finally, transitory silencing of RXRα and RXRβ single isoforms during the first stages of RA-mediated differentiation distinctly affected cellular phenomena induced by RA: whereas RXRα is required for genomic and non-genomic effects during differentiation, RXRβ negatively regulate neurite extension in SH-SY5Y cells. Results thus indicate distinct functions for RXR isoforms during the first stages of RA-dependent neuronal differentiation of neuroblastoma, and reveal new perspectives for studying RXRs as molecular targets in neuronal replacement therapies.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of requirements for the degree of Master of Science. Johannesburg, 2015. / Over the decades, cancer has become a global burden with alarmingly high incidence and mortality rates in both economically developed and developing countries. Characteristically, tumour cells exhibit an over-expression of the 37kDa/67kDa laminin receptor (LRP/LR) in comparison to their normal cell counterparts, with this receptor being implicated in several tumourigenic processes – importantly for the present study, the maintenance of cellular viability and the evasion of apoptosis. This present study aimed to elucidate the role of LRP/LR on the cellular viability of pancreatic cancer (AsPC-1) and neuroblastoma (IMR-32) cells. Flow cytometry revealed that both of these tumourigenic cell lines exhibited LRP/LR on their surface, with further analysis using median fluorescence intensity values showing that IMR-32 cells contain about 70% more cell-surface LRP/LR than AsPC-1 cells. Additionally, Western blotting and densitometry suggested that IMR-32 cells contained about 63% more total LRP/LR than AsPC-1 cells. Western blot analysis also revealed that targeting the mRNA of the 37kDa LRP using a LRP-specific siRNA (siRNA-LAMR1) in AsPC-1 and IMR-32 cells led to significant down-regulation of 90% and 71% in LRP expression, respectively. Consequently, MTT assays showed that LRP knockdown led to reductions of 82% and 65% in the viability of AsPC-1 and IMR-32 cells, respectively. Moreover, use of an alternative LRP-specific siRNA (esiRNA-RPSA) confirmed the specificity and excluded an off-target effect of siRNA-LAMR1 for LRP. BrdU assays revealed that knockdown of LRP reduced the proliferation of AsPC-1 and IMR-32 cells by 76% and 44%, respectively. Confocal microscopy indicated nuclear morphological changes suggestive of apoptosis as the form of cell death occuring in both cell lines after LRP down-regulation. This observation was confirmed using Annexin-V assays, which revealed that AsPC-1 cells underwent 44% more apoptosis than IMR-32 cells post LRP knockdown. Furthermore, caspase-3 activity assays revealed that both cell lines experienced apoptotic induction after siRNA-mediated down-regulation of LRP. Caspase-8 and -9 activity assays suggested that post LRP knockdown, IMR-32 cells undergo apoptosis solely via the extrinsic pathway, whilst AsPC-1 cells use both the intrinsic and extrinsic apoptotic pathways, possibly through a retaliatory feedback loop. Overall, LRP/LR is critical for the maintenance of the tumour cellular viability, making the receptor a promising therapeutic target and proposing the potential use of siRNA technology for treatment of pancreatic cancer and neuroblastoma.

Multidrug resistance (MDR) is one of the foremost causes of treatment failure in childhood malignancies. MDR is a multifactorial process, but classic resistance to cytotoxic drugs has most often been associated with over-expression of one or more MDR transporter proteins in malignant cells, conferring on them the ability to extrude an extraordinarily diverse array of endo- and xenobiotics out of the cell. The best characterized multidrug transporters, P-glycoprotein (Pgp) and the Multidrug Resistance-associated Protein (MRP), belong to the ATP-binding cassette (ABC) gene superfamily, and have been previously implicated in the development of drug resistance in the clinical context. The work described herein examined the various aspects of the MDR genotype and phenotype in the childhood malignancies acute lymphoblastic leukaemia (ALL) and neuroblastoma. The first series of studies tested the hypothesis that morphine, a potential Pgp substrate, might influence the efficacy and/or toxicity of clinically used chemotherapy agents which are substrates for Pgp. The results, however, provided no evidence in a variety of human tumour cell lines of morphine influencing response to selected chemotherapeutic drugs. This finding is particularly important as morphine remains to be the opioid of choice for the treatment of cancer pain in the clinic. The second series of studies examined the effect of single nucleotide polymorphisms (SNPs) in the MDR1 gene, encoding Pgp, and in the MRP1 gene, on patient outcome in childhood ALL or neuroblastoma, with a view to identifying novel prognostic markers for these malignancies. It was found that two of the examined SNPs in the MRP1 gene were associated with improved outcome in neuroblastoma, which had not previously been demonstrated in this disease. Moreover, each of the relevant MRP1 SNPs were associated with lower MRP1 gene expression in both patient samples and tumour cell lines, supporting previous studies indicating that low MRP1 expression in neuroblastoma is strongly associated with improved patient outcome. Importantly, the results of this study suggest a role for selected MRP1 polymorphisms in predicting clinical response in neuroblastoma. Finally, a series of studies were undertaken, using both in vitro and in vivo model systems, to test the efficacy of putative small molecule inhibitors of the MRP1 gene and its transcriptional regulator, the MYCN oncogene, in neuroblastoma. These studies demonstrated for the first time the efficacy of a novel compound, 4H10, at reversing multidrug resistance either in cultured neuroblastoma cells, or in the MYCN transgenic mice, which develop neuroblastoma that closely mirror the human disease. The results indicate that inhibition of MRP1 function has potential clinical importance in the treatment of neuroblastoma, and therefore warrant further research in this area. In contrast, the results failed to provide evidence of the in vivo efficacy of the novel putative small molecule MYCN inhibitors analysed in these studies. Collectively, the findings of these studies contribute to a better understanding of the mechanisms of clinical drug resistance, and may help in the development of new approaches for risk assessment and treatment of aggressive childhood malignancies and thereby improve the long-term outlook of children diagnosed with these debilitating diseases.

Multidrug resistance (MDR) is one of the foremost causes of treatment failure in childhood malignancies. MDR is a multifactorial process, but classic resistance to cytotoxic drugs has most often been associated with over-expression of one or more MDR transporter proteins in malignant cells, conferring on them the ability to extrude an extraordinarily diverse array of endo- and xenobiotics out of the cell. The best characterized multidrug transporters, P-glycoprotein (Pgp) and the Multidrug Resistance-associated Protein (MRP), belong to the ATP-binding cassette (ABC) gene superfamily, and have been previously implicated in the development of drug resistance in the clinical context. The work described herein examined the various aspects of the MDR genotype and phenotype in the childhood malignancies acute lymphoblastic leukaemia (ALL) and neuroblastoma. The first series of studies tested the hypothesis that morphine, a potential Pgp substrate, might influence the efficacy and/or toxicity of clinically used chemotherapy agents which are substrates for Pgp. The results, however, provided no evidence in a variety of human tumour cell lines of morphine influencing response to selected chemotherapeutic drugs. This finding is particularly important as morphine remains to be the opioid of choice for the treatment of cancer pain in the clinic. The second series of studies examined the effect of single nucleotide polymorphisms (SNPs) in the MDR1 gene, encoding Pgp, and in the MRP1 gene, on patient outcome in childhood ALL or neuroblastoma, with a view to identifying novel prognostic markers for these malignancies. It was found that two of the examined SNPs in the MRP1 gene were associated with improved outcome in neuroblastoma, which had not previously been demonstrated in this disease. Moreover, each of the relevant MRP1 SNPs were associated with lower MRP1 gene expression in both patient samples and tumour cell lines, supporting previous studies indicating that low MRP1 expression in neuroblastoma is strongly associated with improved patient outcome. Importantly, the results of this study suggest a role for selected MRP1 polymorphisms in predicting clinical response in neuroblastoma. Finally, a series of studies were undertaken, using both in vitro and in vivo model systems, to test the efficacy of putative small molecule inhibitors of the MRP1 gene and its transcriptional regulator, the MYCN oncogene, in neuroblastoma. These studies demonstrated for the first time the efficacy of a novel compound, 4H10, at reversing multidrug resistance either in cultured neuroblastoma cells, or in the MYCN transgenic mice, which develop neuroblastoma that closely mirror the human disease. The results indicate that inhibition of MRP1 function has potential clinical importance in the treatment of neuroblastoma, and therefore warrant further research in this area. In contrast, the results failed to provide evidence of the in vivo efficacy of the novel putative small molecule MYCN inhibitors analysed in these studies. Collectively, the findings of these studies contribute to a better understanding of the mechanisms of clinical drug resistance, and may help in the development of new approaches for risk assessment and treatment of aggressive childhood malignancies and thereby improve the long-term outlook of children diagnosed with these debilitating diseases.

Neuroblastoma (NB) is the most common extra-cranial solid tumor in children. Recently discovered neuroblastoma tumor-initiating cells (NB-TICs) have many properties of cancer stem cells and form tumors with as few as 10 cells. To elucidate the signaling pathways driving NB- TIC survival and proliferation, we surveyed the phospho-tyrosine containing subset of the NB- TIC proteome. Over 300 phosphorylated proteins were identified, including 21 tyrosine kinases of which several belong to the Src kinase family. Using bioinformatics tools, several hematopoietic signaling pathways were identified, including the B cell receptor (BCR) pathway. Further proteomic approaches substantiated molecular hematopoietic features in NB-TICs. Inhibitors of BCR proximal kinases SYK and SFKs were cytotoxic to NB-TICs. Clinically utilized inhibitors of SFKs induce apoptosis in NB-TICs. Targeting hematopoietic survival pathways in NB-TICs from the bone marrow, which have thus far not been predicted to play a role in this neural malignancy, may provide new drug therapies for NB.

Neuroblastoma (NB) is the most common extra-cranial solid tumor in children. Recently discovered neuroblastoma tumor-initiating cells (NB-TICs) have many properties of cancer stem cells and form tumors with as few as 10 cells. To elucidate the signaling pathways driving NB- TIC survival and proliferation, we surveyed the phospho-tyrosine containing subset of the NB- TIC proteome. Over 300 phosphorylated proteins were identified, including 21 tyrosine kinases of which several belong to the Src kinase family. Using bioinformatics tools, several hematopoietic signaling pathways were identified, including the B cell receptor (BCR) pathway. Further proteomic approaches substantiated molecular hematopoietic features in NB-TICs. Inhibitors of BCR proximal kinases SYK and SFKs were cytotoxic to NB-TICs. Clinically utilized inhibitors of SFKs induce apoptosis in NB-TICs. Targeting hematopoietic survival pathways in NB-TICs from the bone marrow, which have thus far not been predicted to play a role in this neural malignancy, may provide new drug therapies for NB.

Background Mesenchymal stem cells (MSC) were first discovered in the 1970s by scientist A.J. Friedenstein and his colleagues. Friedenstein isolated the first mesenchymal stem cells and was credited for discovering its multilineage differentiation potential. To this day, an extensive amount of research has been conducted on the use of these cells in the treatment of degenerative diseases and various autoimmune disorders. Its migratory ability and immunosuppressive characteristics make MSCs advantageous in an inflammatory environment. Recently, MSCs were also found to have the ability to phagocytose apoptotic bodies generated from T-cells and B-cells. Objectives In this study, we sought to investigate the phagocytic capability of MSCs further in a cancer setting and observe whether or not MSCs could become immunostimulatory cells after phagocytosis of apoptotic cancer cells. Methods To conduct this study, we first used UV-irradiation to generate apoptotic cells from 3 neuroblastoma (NB) cell lines. After apoptotic NB cells were generated, they were then co-cultured with MSCs for phagocytosis to occur. To detect phagocytosis, we stained the apoptotic NB cells with a red fluorescent dye PKH-26 and MSCs with CFSE, a green fluorescent dye. Then, we used flow cytometry to detect the percentage of phagocytosis. After phagocytosis, we collected the supernatants from the MSCs treated with the apoptotic NB cells and observed how the IL-6 and IL-8 cytokine levels changed compared to the levels from the supernatant of the MSCs only. Results and Conclusions After conducting this experiment, our results showed that in a cancer environment MSCs were able to phagocytose apoptotic NB cells. Furthermore, after phagocytosis the IL-6 and IL-8 cytokine levels increased significantly in the MSCs treated with apoptotic NB cells compared to the control group with MSCs only. Since IL-6 and IL-8 are both considered pro-inflammatory cytokines, we can conclude that after phagocytosis of apoptotic NB cells, MSCs can become immunostimulatory cells. To further confirm our findings, various other cytokines should be tested and more experiments should be done. This way, a more complete picture can be generated describing how MSC cytokine secretion activity changes after phagocytosis of apoptotic neuroblastoma cells. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Medical Sciences

Multidrug resistance (MDR) is one of the foremost causes of treatment failure in childhood malignancies. MDR is a multifactorial process, but classic resistance to cytotoxic drugs has most often been associated with over-expression of one or more MDR transporter proteins in malignant cells, conferring on them the ability to extrude an extraordinarily diverse array of endo- and xenobiotics out of the cell. The best characterized multidrug transporters, P-glycoprotein (Pgp) and the Multidrug Resistance-associated Protein (MRP), belong to the ATP-binding cassette (ABC) gene superfamily, and have been previously implicated in the development of drug resistance in the clinical context. The work described herein examined the various aspects of the MDR genotype and phenotype in the childhood malignancies acute lymphoblastic leukaemia (ALL) and neuroblastoma. The first series of studies tested the hypothesis that morphine, a potential Pgp substrate, might influence the efficacy and/or toxicity of clinically used chemotherapy agents which are substrates for Pgp. The results, however, provided no evidence in a variety of human tumour cell lines of morphine influencing response to selected chemotherapeutic drugs. This finding is particularly important as morphine remains to be the opioid of choice for the treatment of cancer pain in the clinic. The second series of studies examined the effect of single nucleotide polymorphisms (SNPs) in the MDR1 gene, encoding Pgp, and in the MRP1 gene, on patient outcome in childhood ALL or neuroblastoma, with a view to identifying novel prognostic markers for these malignancies. It was found that two of the examined SNPs in the MRP1 gene were associated with improved outcome in neuroblastoma, which had not previously been demonstrated in this disease. Moreover, each of the relevant MRP1 SNPs were associated with lower MRP1 gene expression in both patient samples and tumour cell lines, supporting previous studies indicating that low MRP1 expression in neuroblastoma is strongly associated with improved patient outcome. Importantly, the results of this study suggest a role for selected MRP1 polymorphisms in predicting clinical response in neuroblastoma. Finally, a series of studies were undertaken, using both in vitro and in vivo model systems, to test the efficacy of putative small molecule inhibitors of the MRP1 gene and its transcriptional regulator, the MYCN oncogene, in neuroblastoma. These studies demonstrated for the first time the efficacy of a novel compound, 4H10, at reversing multidrug resistance either in cultured neuroblastoma cells, or in the MYCN transgenic mice, which develop neuroblastoma that closely mirror the human disease. The results indicate that inhibition of MRP1 function has potential clinical importance in the treatment of neuroblastoma, and therefore warrant further research in this area. In contrast, the results failed to provide evidence of the in vivo efficacy of the novel putative small molecule MYCN inhibitors analysed in these studies. Collectively, the findings of these studies contribute to a better understanding of the mechanisms of clinical drug resistance, and may help in the development of new approaches for risk assessment and treatment of aggressive childhood malignancies and thereby improve the long-term outlook of children diagnosed with these debilitating diseases.