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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

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Indiana University-Purdue University (IUPUI) / Most pancreatic cancer patients receiving gemcitabine chemotherapy eventually develop resistance to gemcitabine. To improve survival and prognosis of pancreatic cancer patients, better understanding the mechanisms of gemcitabine resistance and discovery of new therapeutic targets are required. In this study, I investigated the molecular mechanisms of acquired gemcitabine resistance using a stepwise gemcitabine-selected pancreatic cancer cell line in comparison to the parental cell line. I found that 14-3-3σ is up-regulated in the drug resistant cell line due to demethylation in its first exon, and the up-regulation of 14-3-3σ gene expression, in turn, contributes to gemcitabine resistance. Intriguingly, I found that demethylation of the 14-3-3σ gene in gemcitabine resistant cells is reversibly regulated by DNMT1 and UHRF1. Furthermore, I found that 14-3-3σ over-expression causes gemcitabine resistance by inhibiting gemcitabine-induced apoptosis and caspase-8 activation possibly via binding to YAP1. The finding of demethylation of the 14-3-3σ gene in gemcitabine resistant cells led to a hypothesis that other genes may also be changed epigenetically following gemcitabine selection. By RRBS (Reduced Representation Bisulfite Sequencing) analysis, 845 genes were found to have altered methylation. One of these genes, PDGFD, was further investigated and found to have reversible demethylation at its promoter region in the drug resistant cells and contribute to gemcitabine resistance possibly via autocrine activation of the STAT3 signaling pathway. Together, these findings not only provide evidence that 14-3-3σ and PDGFD over-expression contribute to acquired gemcitabine resistance and that reversible epigenetic changes may play an important role in acquired gemcitabine resistance, but also demonstrate that the molecular mechanisms of acquired gemcitabine resistance in pancreatic cancer cells are complex and multifaceted.

Indiana University-Purdue University Indianapolis (IUPUI) / Pancreatic ductal adenocarcinoma (PDAC), which comprises 85% of pancreatic cancers, is the 4th leading cause of cancer death in the United States with a 5-year survival rate of 8%. While human PDACs (hPDACs) are hypovascular, they also overexpress a number of angiogenic growth factors and receptors. Additionally, the use of anti-angiogenic agents in murine models of PDAC leads to reduced tumor volume, tumor spread, and microvessel density (MVD), and improved survival. Nonetheless, clinical trials using anti-angiogenic therapy have been overwhelmingly unsuccessful in hPDAC. On the other hand, pancreatic neuroendocrine tumors (PNETs) account for only 2% of pancreatic tumors, yet they are very vascular and classically angiogenic, respond to anti-angiogenic therapy, and confer a better prognosis than PDAC even in the metastatic setting. In an effort to compare and contrast the angiogenic transcriptomes of these two tumor types, we analyzed RNA-Sequencing (RNA-Seq) data from The Cancer Genome Atlas (TCGA) and found that a pro-angiogenic gene signature is present in 35% of PDACs and that it is mostly distinct from the angiogenic signature present in PNETs. The pro-angiogenic PDAC subgroup also exhibits a transcriptome that reflects active TGF-β signaling, less frequent SMAD4 inactivation than PDACs without the signature, and up-regulation of several pro-inflammatory genes, including members of JAK signaling pathways. Consequently, targeting the TGF-β receptor type-1 kinase with SB505124 and JAK1/2 with ruxolitinib blocks proliferative crosstalk between human pancreatic cancer cells (PCCs) and human endothelial cells (ECs). Additionally, treatment of the KRC (oncogenic Kras, homozygous deletion of Rb1) and KPC (oncogenic Kras, mutated Trp53) genetically engineered PDAC mouse models with ruxolitinib suppresses murine PDAC (mPDAC) progression only in the KRC model, which shows superior enrichment and differential expression of the human pro-angiogenic gene signature as compared to KPC tumors. These findings suggest that targeting both TGF-β and JAK signaling in the 35% of PDAC patients whose cancers exhibit an pro-angiogenic gene signature should be explored in a clinical trial.

Indiana University-Purdue University Indianapolis (IUPUI). / The abysmal five year pancreatic cancer survival rate of less than 6% highlights the need for new treatments for this deadly malignancy. Cytotoxic drugs normally target rapidly dividing cancer cells but unfortunately often target stem cells resulting in toxicity. This warrants the development of compounds that selectively target tumor cells. An inhibitor of the mitotic kinase, MPS1, which has been shown to be more selective towards cancer cells than non-tumorigenic cells, shows promise but its effects on stem cells has not been investigated. MPS1 is an essential component of the Spindle Assembly Checkpoint and is proposed to be up-regulated in cancer cells to maintain chromosomal segregation errors within survivable limits. Inhibition of MPS1 kinase causes cancer cell death accompanied by massive aneuploidy. Our studies demonstrate that human adipose stem cells (ASCs) and can tolerate higher levels of a small molecule MPS1 inhibitor than pancreatic cancer cells. In contrast to PANC-1 cancer cells, ASCs and telomerase-immortalized pancreatic ductal epithelial cells did not exhibit elevated chromosome mis-segregation after treatment with the MPS1 inhibitor for 72hrs. In contrast, PANC-1 pancreatic cancer cells exhibited a large increase in chromosomal mis-segregation under similar conditions. Furthermore, growth of ASCs was minimally affected post treatment whereas PANC-1 cells were severely growth impaired suggesting a favorable therapeutic index. Our studies, demonstrate that MPS1 inhibition is selective towards pancreatic cancer cells and that stem cells are less affected in vitro. These data suggest MPS1 inhibition should be further investigated as a new treatment approach in pancreatic cancer.