A dissertation submitted to the Faculty of Science, University of Witwatersrand, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2014.

Angiogenesis is the formation of new vessels from existing vasculature and is essential for tumour growth and metastasis. It is controlled by angiogenic factors secreted by the tumour which regulate the matrix remodelling, endothelial cell (EC) proliferation, and capillary differentiation necessary for establishing a blood supply. This thesis has examined angiogenesis in human tumours. Immunohistochemically highlighted vessels in tumours were quantified using different methods to develop a rapid and objective method for measuring tumour angiogenesis. Significant associations between Chalkley counting, microvessel density, vascular area and perimeter were demonstrated; the Chalkley technique gave independent prognostic information and was suitable for a diagnostic service. Studies on the frequency EC of S-phase showed proliferation (labelling index 2.2%) occurs mostly at the tumour margin suggesting the growth factors controlling ECs are different from those regulating tumour cells and that remodelling the existing vasculature might play a more important role than previously recognised. To investigate further ECtumour matrix interactions, the expression of cell adhesion molecules (CAMs) was examined. CAM expression mirrored that of EC proliferation with preferential expression on endothelium at the tumour periphery: expression of CAMs was also present on neoplastic cells. Thus, acquisition of CAMs by tumour cells together with EC phenotypic modulation might promote angiogenesis and metastasis. The angiogenic factor thymidine phosphorylase (TP) was examined in normal tissues and tumours. Although TP was expressed in ECs there was no correlation between expression in normal or neoplastic tissue and vascularity. Nevertheless, TP was elevated in small low grade tumours, in accordance with TP being chemotactic but non-mitogenic for ECs. A monoclonal antibody to flt-4, a candidate angiogenic factor receptor was generated and characterised. In contrast to the in-situ mRNA expression profile, a restricted pattern of protein expression was observed in normal tissues and variable expression in tumours.

This thesis examined the effect and biochemical mechanism by which combined vitamin E and C supplementation may influence tumour cell growth. The study initially addressed the effect of combined vitamin E succinate and Asc supplementation over a nutritional concentration range (5- 20μg/ml) and (25-50μg/ml) respectively, on the in vitro growth of non-malignant LLCMK and malignant BL6 cells. Supplementation of BL6 and LLCMK cells with combined vitamin E succinate and ascorbic acid, resulted in no significant increasing or decreasing trend in LLCMK cell growth, while in BL6 cells a significant decrease in cell growth was observed at all combined vitamin concentrations. It has been suggested that these vitamins may act synergistically to inhibit tumour cell growth through their antioxidant properties in quenching free radicals and lipid peroxidation and furthermore through their modulation of the activities of various enzymes and metabolites in the eicosanoid pathway. This study consequently investigated the effects of combined vitamin E succinate and ascorbic acid supplementation on these parameters. Throughout this study, emphasis was placed on the BL6 melanoma cells, as combined vitamin E succinate and ascorbic acid supplementation did not significantly affect growth or levels of secondary metabolites in the non-malignant LLCMK cells. Combined vitamin E succinate and ascorbic acid supplementation of BL6 cells resulted in a marked but non significant increase in free radical and a significant increase in lipid peroxidation levels. This prooxidant effect was accompanied by a significant decrease in BL6 cell growth, suggesting that the growth inhibitory effects of combined vitainin E succinate and ascorbic acid on BL6 cells in vitro was not mediated through their synergistic antioxidant properties. Vitamin E succinate is a nonphysiological antioxidant in its esterified form, hence cleavage of the succinate group must occur in order for ascorbic acid to interact with the free alcohol, vitamin E. The inability of combined vitamin E succinate and ascorbic acid to reduce free radicals and lipid peroxidation levels within BL6 cells may not be due to their ineffectiveness as antioxidants but rather the presence of other contributing factors which influence the oxidation state within the BL6 cells. Vitamin E is believed to modulate membrane-bound enzymes through membrane stabilization. Furthermore, the stabilizing effect of vitamin E may be enhanced by the ascorbic acid-sparing effect of vitamin E. Hence, this study investigated the effect of combined vitamin E succinate and ascorbic acid in modulating the activity of various enzymes and secondary messengers in the eicosanoid pathway. Supplementation with combined vitamin E succinate (5-20μg/ml) and ascorbic acid (25-50μg/ml) resulted in significant increases in phospholipase A₂, 5-lipoxygenase, cyclooxygenase and adenyl ate cyclase activity, with a significant decrease in BL6 cell growth. The possible synergistic action of these vitamins in terms of modulating membrane-bound enzymes was further substantiated by uptake and cellular distribution studies. Vitamin E succinate and vitamin E in the membrane fraction increased significantly compared to control cultures, while ascorbic acid levels were significantly higher in the stroma fraction when compared to membrane fractions. Consequently, another factor accounting for increased activities of phospholipase A2, 5-lipoxygenase and adenylate cyclase activities as a result of vitamin supplementation in BL6 cells may be an increased availability of Ca²+. Supplementation of BL6 cells with combined vitamin E succinate and ascorbic acid resulted in significant increases in intracellular Ca²+ levels at all combined vitamin groups. Furthermore, this increase in intracellular Ca²+ was positively correlated with cl1anges of the above-mentioned enzyme activities. Within the eicosanoid pathway, the rate of prostaglandin synthesis is regulated by phospholipase A₂ activity and arachidonic acid release, and the net prostaglandin production is dependent on cyclooxygenase activity, hence the effects of combined vitamin E succinate and ascorbic acid on arachidonic acid composition and prostaglandin production within BL6 cells was determined. The percentage arachidonic acid composition of the BL6 cells was elevated and inversely related to cell growth following combined vitamin E succinate and ascorbic acid supplementation. Prostaglandin E₂ and prostaglandin I₂ levels increased significantly, while those of prostaglandin D2 and prostaglandin F₂α increased markedly following supplementation of combined vitamin E succinate and ascorbic acid. These increases in prostaglandin levels were inversely related to BL6 cell growth, suggesting that the prostaglandins were involved in negative regulation of BL6 cell growth. When comparing the levels of prostaglandins, prostaglandin E2 levels were significantly higher when compared to prostaglandin D₂, prostaglandin F₂α and prostaglandin I₂ suggesting that vitamin E₂ succinate and ascorbic acid effects were mediated primarily through an increase in prostaglandin E2. Hence, prostaglandin E2 levels in combined vitamin E succinate and ascorbic acid appeared to be dependent on the amount of precursor present and the activity of its synthetic enzymes. This was confirmed when BL6 cells were supplemented with arachidonic acid. Arachidonic acid had an inhibitory effect on BL6 cell growth and also stimulated prostaglandin E₂ production. Prostaglandin E₂ levels are in turn believed to modulate adenylate cyclase activity in BL6 cells, hence it is reasonable to conclude that adenylate cyclase activity is dependent on prostaglandin E₂ levels. Combined vitamin E succinate and Asc supplementation to BL6 cells resulted in significant increases in adenyl ate cyclase and cyclic adenosine monophosphate, which again correlated with a significant decrease in cell growth. As cyclic adenosine monophosphate has a regulatory role in the cell cycle this study suggested that the effect of combined vitamin E succinate and ascorbic acid supplementation was mediated through the final effect provided by the second messenger, cyclic adenosine monophosphate. This was confirmed when BL6 cells were supplemented with dexamethasone, a phospholipase A₂ inhibitor. This treatment rsulted in combined vitamin E succinate and ascorbic acid having no inhibitory effect on BL6 cell growth. Cyclooxygenase activity, prostaglandin E₂ levels, adenylate cyclase activity and cyclic adenosine monophosphate levels were significantly lower in dexamethasone-treated cells compared to non-treated dexamethasone cultures. The reason for the increased free radical and lipid peroxidation levels in BL6 cells was further investigated. Cyclooxygenase enzymes are believed to generate free radical species during catalytic activity. Analysis of free radical and lipid peroxidation levels following supplementation with dexamethasone revealed markedly lower free radical and significantly lower lipid peroxidation levels in comparison with control cultures and non dexamethasone-treated cultures. These results suggest that the observed increases in free radical and lipid peroxidation levels in BL6 cells supplemented with combined vitamin E succinate and ascorbic acid were indirectly due to the increase in cyclooxygenase activity in these cells.

published_or_final_version / abstract / Pharmacology / Doctoral / Doctor of Philosophy

Phospholipase C mediated hydrolysis of the phosphoinositides yields second messenger products which have been linked to normal and abnormal cell growth processes. It has been suggested that fatty acids may regulate phospholipase C activity in vivo. This study sought to investigate relationships between phospholipase C activity, tumor size and dietary linoleic and stearic acids.Phospholipase C activity, of livers and serially transplanted mammary adenocarcinomas from female strain A/ST mice on one of three diets, was measured. Tumors ranged from .20 to 2.98 g at the time of removal. Phospholipase C activity of tumors was negatively and significantly (p<.Ol) correlated to increasing tumor weight in those diet groups that contained linoleic acid and no added stearic acid. The highest PLC activity was seen in small tumors across all diet groups. Maximum PLC activity of tumors was over 2 fold higher than the maximum activity of livers across all diet groups. PLC activity of the liver was negatively and significantly (p<.02) correlated to increasing tumor weight for samples from the high linoleic acid diet (SF-15). This research concludes that PLC activity varies during tumorigenesis and may reflect tumor development. / Department of Biology

Macrophages (Mφ) are a major immune cell involved in anti-tumor responses. Mφ activities such as tumor cytotoxicity. presentation of tumor-associated antigens, and stimulation of anti-tumor lymphocytes are all involved in the battle against tumor growth. However, other Mφ activities such as cell growth promotion, angiogenesis, and suppression of anti-tumor lymphocytes aid in tumor growth. This dissertation discusses how tumors control Mφ activities to create favorable environments for tumor growth. Assessment of tumor- and Mφ-derived molecules has enabled me to design models of communication between tumors, Mφ, and other immune cells. A major research focus was to determine how tumor-derived molecules induce Mφ suppressor activity and control Mφ cytotoxicity. Tumor growth induced Mφ to suppress T lymphocyte proliferation by increasing Mφ production of the suppressor molecules prostaglandin E₂ (PGE₂), nitric oxide (NO), and tumor necrosis factor-α (TNF-α). A major finding was that TNF-α's normal up-regulatory action on T-cell proliferation switched to a suppressor action when Mφ were present. The autocrine action of increased TNF-α levels during tumor growth stimulated Mφ PGE₂ and NO synthesis, which suppressed T-cell proliferation. / Ph. D.

The type 1 insulin-like growth factor receptor (lGF-1 R) plays a significant role in tumor growth and spread, and IGF-1 R inhibitors and antibodies are now undergoing clinical testing. However, factors that regulate sensitivity to IGF-1 R inhibition remain unclear. The aim of this project is to identify proteins whose depletion regulates sensitivity to IGF-1 R inhibition, in order to design effective combination treatments to benefit patients. An IGF-1 R kinase inhibitor, AZ12253801 (provided by AstraZeneca) was able to block IGF-induced phosphorylation of IGF-1 R in DU145 prostate cancer and MCF-7 breast cancer cells, inhibited downstream signalling in DU145 cells, and also inhibited proliferation and cell survival of both cell lines. AZ12253801 was used in an unbiased siRNA screen in both cell lines, using two s'iRNA libraries (779 kinase-related Kinome and 230 DNA repair-associated siRNAs). Eight Kinome and five DNA repair-associated hits have been identified after primary and second round screens, and further validated. The strongest hit was dishevelled homolog 3 (DVL3), a member of the WNT signalling pathway, which is highly expressed in both cell lines. DVL3 silencing caused reduction in active l3-catenin and inactivated the mTOR pathway, consistent with previous studies, and did not affect IGF-1 Rand AKT activity. However, DVL3 silencing led to activation of MEK1/2-ERK1/2 in serum-starved cells and sensitized this pathway to IGF-1 stimulation, with translocation of ERK1/2 into the nucleus and increased expression of ERK1/2 target genes. A DVL PDZ domain inhibitor (DVLi) showed similar effects on active l3-catenin, mTOR signalling and ERK1/2 signalling activity. The administration of DVLi increased sensitivity to AZ12253801 in cell lines with detectable ERK1/2 activation, but not prostate cancer cells in which ERK signalling was suppressed and AKT was activated in the context of loss of functional PTEN. Furthermore, DVL3 regulated activation of ERKs by influencing signaling downstream of the IGF-1 R and upstream of RAS, and DVL3 was found in a complex with the adaptor proteins GRB2 and DAB2. GRB2 knockdown was capable of abolishing ERK1/2 activation induced by DVLi, further implicating involvement of GRB2, and DAB2 silencing sensitized to IGF-1 R inhibition, mimicking effects of DVL3 depletion. Taken together, DVL3 silencing or inhibition enhances sensitivity to IGF-1 R inhibition by negatively regulating the ERK1/2 signaling pathway. These investigations shed new light on the factors that regulate IGF signaling, and provide a rational basis for design of clinical trials of IGF-1 R inhibitors.

The spontaneous tumoricidal ability of pulmonary alveolar macrophages (PAM) isolated from C57B1/6 mice bearing either a metastatic or a nonmetastatic cloned variant of Lewis lung carcinoma (LLC) was examined in vitro. During the early weeks of tumor development the cytotoxicity mediated by macrophages was enhanced in the tumor-bearing mice, especially in the metastatic tumor bearers. Later in tumor progress (week 4) the spontaneous cytotoxicity of both groups typically declined to levels less than those of normal macrophages. Experiments were performed to determine if macrophages could be activated further in vitro by incubation in a mixture of lymphokine and lipopolysaccha ride. The macrophages from the metastatic tumor bearers were consistently activated in vitro. However, macrophages isolated from mice bearing large tumors and whose spontaneous cytotoxicity was suppressed could not be activated.The secretion of prostaglandin E2 (PGE2) by macrophages at different times during tumor development was measured to determine if PGE2 levels corresponded with the ability or inability of macrophages to kill tumor cells. Secretion of PGE2 typically corresponded with the capacity to kill rather than with an inability to kill target cells. Similarly, the production of PGE2 by macrophages was not responsible for the decline in the ability of macrophages to kill tumor cells.These results suggest that PAM are activated to be cytotoxic during the period when pulmonary metastases are developing. The successful establishment of these metastases does not appear to depend on the capacity of the tumor to suppress alveolar macrophage cytotoxicity. / Department of Biology

Notch signalling plays a key role in physiological development and tumourigenesis. The recent discovery and characterisation of Notch ligand Delta-like 4 (D114), which is predominantly expressed in endothelial cells, have underscored the role of Notch signalling in angiogenesis. This thesis investigates the regulation and function of D114-Notch signalling in angiogenesis and tumourigenesis. First, the D114-Notch pathway interacted with the cellular hypoxia-sensing pathway. In human umbilical vein endothelial cells (HUVECs), D114 overexpression repressed hypoxic induction, and the repression was mediated by Notch target gene Hey2. In the breast cancer cell line MCF7, hypoxia induced Notch target gene Hey1 and ligand Jagged2 via hypoxia inducible factor 1. The hypoxic induction of Hey1 was also dependent on Notch signalling. Second, D114 expression in HUVECs was up-regulated by several pathways. Notch signalling, activated by receptor overexpression, ligand stimulation or cell-cell contact, induced D114 expression. Treatment with vascular endothelial growth factor (VEGF) or hypoxia also induced D114 expression via Notch signalling. In addition, VEGF promoted Notch signalling and D114 expression in tumours. Third, D114 expression in HUVECs was up-regulated by co-culturing with cancer cells. B16 mouse melanoma cells and human breast cancer cell lines induced D114 expression in HUVECs via Notch signalling. B16 cells also induced D114 expression via soluble factors independent of Notch signalling. Finally, D114-Notch signalling regulated tumour growth in vivo. D114 overexpression in cancer cell lines activated Notch signalling in the stroma of xenograft tumours, and promoted the growth of human U87 (glioblastoma) and PC3 (prostate cancer) xenografts. In addition, D114-overexpressing U87 tumours were resistant to anti- VEGF treatment during later stages; however, they did respond to anti-Notch treatment. Altogether, the D114-Notch pathway is tightly regulated and plays an important role in physiological and tumour angiogenesis. Inhibiting this pathway may be a viable therapy for cancers resistant to VEGF inhibition.

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