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A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements of the degree of Masters of Science. February 2017. / Cancer has become a problematic fatal disease in developing and industrialised countries with pancreatic cancer as the seventh leading cause of cancer-related deaths, with an average survival rate of less than 5%. Environmental risk factors associated with pancreatic cancer include smoking, obesity, diet, alcohol etc. Furthermore, pancreatic cancer is commonly diagnosed at a late stage where its response to current anti-cancer agents is poor. Consequently, with South Africa being a 3rd world country and the cost of chemotherapy being so high, this has led to us trying to identify new, cheaper therapeutics for cancer cells. A majority (80%) of the South African population relies on traditional medicines, hence in this study we aimed to assess Lobostemon fruticosus for anti-proliferative effects on pancreatic cancer cell line (AsPC-1). This was achieved by the use of methanol and butanol extracts of L. fruticosus to screen for induction of apoptosis and inhibition of cell proliferation. The plant was collected, dried, crushed and dissolved in butanol and methanol to obtain experimental extracts. Cytotoxicity of the plant on Aspc-1 was determined using MTT Assay, xCELLigence and cell cycle analysis. MRC-5 cell line was used as a positive control cell line. L. fruticosus extracts induced cell death at IC50 of 60µg/ml (methanol extract) and 50µg/ml (butanol extract) at 48hour treatments on AsPC-1 cell line. Western Blots showed that the methanol and butanol extracts of L. fruticosus led to slight upregulation of the apoptotic gene p53 in AsPC-1 cell line, which was further confirmed by FACS apoptosis detection. Cell cycle analysis further showed the plant extracts do promote cell cycle arrest. LC/MS of the extracts gave spectra of active compounds presumed to play a role in induction of apoptosis on the pancreatic cancer cell line. The data obtained implies that the methanol and butanol extracts of L. fruticosus does have, to a certain extent, growth inhibiting and apoptosis inducing potential on the pancreatic cancer cell line. KEYWORDS: Lobostemon fruticosus, Pancreatic Cancer, methanol extract, butanol extract, AsPC-1 / LG2017

Cell therapy involving treatment of diseases with the body’s own cells would benefit both liver diseases and Type 1 diabetes. Liver diseases are associated with a marked reduction in hepatocytes whilst Type 1 diabetes is characterized by the loss of functional insulin-producing β-cells. Treatment is currently achieved by whole organ liver (or hepatocyte) and islet transplantation methods respectively. However the major limitation to this approach is the shortage of organ donors, thus alternative sources of cells must be found. Potential sources with enormous therapeutic potential are existing cells in the liver and pancreas involved during the regeneration process. In vivo studies have shown progenitor oval cells differentiate into hepatocytes during liver regeneration and α-cells transdifferentiate into β-cells during pancreas regeneration. However neither can be fully exploited until the molecular mechanisms governing their proliferation and trans/differentiation are fully elucidated. Herein we characterise two in vitro cell models, a mouse adult oval cell line, known as BMOL-TAT1.1, and mouse adult pancreatic α-cell line, known as α-TC19 by RT-PCR and immunofluorescent staining. We found that under proliferating culture conditions BMOL-TAT1.1 were heterogenous consisting of two distinct cell types with different β-catenin signalling pathway activation. Inducible differentiation (dexamethasone) induced hepatic and non-hepatic markers in specific cell subtypes, indicating multi-potentiality. Ectopic expression of transcription factor HNF4α in homogenous small BMOL-TAT1.1 cells revealed no hepatic differentiation but potent expression of intestinal markers (Villin, ALPi, ApoAIV). HNF4α was identified as a candidate transcriptional regulator in α- to β-cell transdifferentiation, as ectopic expression in α-TC19 cells, suppressed glucagon and induced expression of several functionally important β-cell markers (GLUT2, GCK, insulin). The contribution of chromatin histone acetylation was also assessed, due to its importance in endocrine fate regulation. In toto these results have important implications for the development of potential therapies to treat liver diseases and Type 1 diabetes.

O Diabetes mellitus tipo 1 (DM1), na maioria dos casos, é causado pela destruição de células β pancreáticas, levando à hiperglicemia. Atualmente a única fonte de novas células β e os únicos tratamentos capazes de restaurar o padrão fisiológico de secreção de insulina nesses pacientes são o transplante de pâncreas e de ilhotas pancreáticas. O transplante de ilhotas apresenta problemas relacionados a enxertia, devido principalmente a baixa vascularização, o que leva à morte de células β nos primeiros dias pós-transplante. Células-tronco mesenquimais apresentam características interessantes para o tratamento do DM1. A primeira aplicação explorada nesse trabalho foi a capacidade de diferenciação de MSCs humanas e murinas em células produtoras de insulina (CPIs). A identidade das células isoladas foi confirmada pela caracterização imunofenotípica e pela capacidade de diferenciação adipogênica e osteogênica in vitro. Quatro protocolos de diferenciação em CPIs foram testados em MSCs derivadas de ilhotas pancreáticas e um em MSCs derivadas de rim murino. A análise da expressão gênica de insulina em células diferenciadas em todos os protocolos testados mostrou níveis insignificantes ou nulos de expressão desse hormônio. A segunda aplicação explorou o co-transplante de ilhotas pancreáticas com MSCs derivadas de rim em camundongos diabéticos. Os resultados mostraram aumento da taxa de cura e melhora na glicemia pós-transplante, bem como uma tendência ao aumento do conteúdo total de insulina em animais co-transplantados em comparação com animais que receberam apenas ilhotas. Não houve diferenças no peso e teste de tolerância à glicose entre os grupos. Foi observado aumento na vascularização do enxerto nos animais que receberam MSCs. Paralelamente, foi estudada a associação de variantes alélicas dos genes PTPN22, KIR, HLA classe I e II e a susceptibilidade ao desenvolvimento de DM1 em uma população do Rio Grande do Sul. Foi observada associação entre o alelo 1858T e o risco aumentado de DM1. A genotipagem do KIR e HLA-C mostrou uma frequência maior de alelos do grupo 2 do HLA-C em controles não diabéticos, bem como o genótipo 2DL1/C2+, sugerindo um papel protetor desse genótipo. Além disso, indivíduos com haplótipo KIR2DL2/DR3+ e KIR2DL2/DR3/DR4+ tem risco aumentado de desenvolvimento de DM1. MSCs parecem possuir baixa capacidade de diferenciação em células β in vitro, entretanto, possuem efeitos benéficos importantes quando cotransplantadas com ilhotas pancreáticas. Essa aplicação tem grande potencial e deveria ser testada em estudos clínicos com o objetivo de melhorar a enxertia e diminuir o número de ilhotas necessárias para cada paciente. / Type 1 Diabetes (DM1), in almost all the cases, is caused by the destruction of beta-cells by cells of the immune system, leading to hyperglycemia. The only source for new beta-cells available is through the pancreas and islet transplantation, two treatments able to restore insulin secretion pattern in this patients. Islet transplantation presents issues related to grafting, caused mainly by poor vascularisation post-transplant, leading to betacell death in the first days after transplantation. Mesenchymal stem cells have interesting characteristics to the treatment of DM1. The first application explored in this work was testing the capacity of differentiation of human and mouse MSCs into insulin-producing cells (CPIs). Identity of isolated cells was confirmed by immunophenotyping and potential of adipogenic and osteogenic differentiation in vitro. Four protocols were tested in human islet-derived MSCs and one in mouse kidney-derived MSCs to generate CPIs. Analysis of insulin expression in differentiated cells from all protocols showed no or very little expression levels of this hormone. The second application was to evaluate the role of MSCs in the co-transplantation with pancreatic islets in diabetes mice. Our results showed an increased number of cured mice and a decrease in glycemic levels post transplant in islet+MSCs group, as well as a tendency to an increase in total insulin content in islet+MSCs compared with islet-only group. No differences could be found in weight and intraperitoneal glucose tolerance test between groups. An increase in graft vascularisation was observed in MSCs-receiving animals. At the same time, we studied the association of allelic variants in PTPN22, KIR, HLA class I and II genes and its association with the developing of DM1. We reported and association of the 1858T allele and an increased risk of DM1. Genotyping shows an increased frequency of group 2 alleles (C2) of HLA-C in controls as well as the 2DL1/C2+ genotype, suggesting a protective role of this genotype. Moreover, individuals with KIR2DL2/DR3+ and KIR2DL2/DR3/DR4+ haplotypes have increased risk of developing DM1. MSCs seem to have low capacity of in vitro differentiation in a beta-cell phenotype, however, they exert important benefic effects when co-transplanted with pancreatic islets in diabetic mice. This application has great potential and should be tested in clinical trials aiming the improvement of islet grafting and decrease in the number of islets needed for transplantation.

Cancer cell metabolism is reorganized around the needs of proliferating cells, particularly the management of organic metabolites and the balance of redox state. Here, we show that pancreatic cancer requires exogenous sources of cysteine for tumor growth and maintenance due to its critical role in redox balance. Using a multidisciplinary approach, we find that cancer cells rely on imported cystine (oxidized cysteine) to detoxify lipid reactive oxygen species (ROS) and avert ferroptosis, a form of non-apoptotic cell death. Cystine–derived glutathione was necessary for this protection, but its depletion was not sufficient to induce ferroptosis. Correspondingly, genetic inactivation of system xc–, the cystine/glutamate antiporter, in established pancreatic tumors induced stabilization or regression, extending survival in an autochthonous mouse model. We observed distinctive lesions of non-apoptotic cell death that may represent an in vivo manifestation of ferroptosis, highlighting a novel, cancer-specific dependency on a potentially druggable membrane channel.

Similar to many cancers, pancreatic cancer (PDA) assumes an altered metabolic state that is designed to support the manufacture of macromolecules necessary for cell division. For example, membrane synthesis from lipid precursors is dependent on de novo fatty acid synthesis (dnFAS), a pathway that is upregulated in many cancers. The rate-limiting enzyme of dnFAS is fatty acid synthase (FASN), an enzyme that is overexpressed in cancer but found at very low levels in normal tissues. Here we demonstrate that fatty acid synthase (FASN) inhibition using novel small molecule inhibitor IPI-9119 induces apoptosis in FASN overexpressing epithelial cells of a pre-clinical pancreatic cancer mouse model (KPC). We also provide evidence that FASN inhibition insensitivity in PDA cells is due to flux through scavenging pathways, particularly macropinocytosis. We initially hypothesized that FASN overexpression is a survival strategy for PDA epithelial cells through which they acclimate to a low nutrient microenvironment. Indeed, K-ras driven metabolic reprograming has been shown to increase flux through the dnFAS pathway indicating that PDA cells should rely heavily on FASN activity. Surprisingly we found that PDA cell lines are minimally affected by FASN inhibition and rely chiefly on scavenging pathways. In vitro cells displayed a dependency on lysosomal function, determined using lysosomal inhibitor chloroquine (CQ). Dual therapy with FASN inhibitor, IPI-9119, and lysosomal inhibitor, CQ, increased apoptosis and decreased cell viability in human PDA cell lines and in bulk KPfl/flC PDA tumors. Our findings suggest that mutated epithelial tumor cells overexpress FASN and that these oncogenic cells can be specifically targeted using FASN inhibition. Our findings in regards to scavenging pathways demonstrate that PDA is reliant on lysosomal activity and may utilize both autophagy and macropinocytosis to maintain lipid levels in addition to dnFAS. Dual therapy with a FASN inhibitor and a lysosomal inhibitor induces apoptosis in PDA cell lines regardless of their sensitivity to FASN inhibition alone indicating that this therapeutic strategy, dual treatment with lysosomal inhibitors and FASN inhibitors, should be further developed. Our major conclusions from this work are: (1) that PDA is not solely dependent on FASN activity for lipid maintenance but short-term inhibition results in targeted apoptosis in FASN expressing cells, in vivo. (2) Lipid scavenging pathway macropinocytosis accounts for primary resistance to FASN inhibition in vitro, although autophagy may also contribute. (3) Dual inhibition of FASN and lysosomal function induces apoptosis and decreases proliferation in PDA cell lines and tumors.