Despite considerable research on cancer treatments and preventatives, poor outcomes in cancer patients are common. The vital search for effective cancer drugs often begins in the laboratory, where unfortunately the effects of a drug in humans cannot be perfectly modelled. Epidemiology can play a vital role in determining the real world efficacy of a drug currently used for other purposes before clinical trials begin. This thesis therefore used primarily laboratory evidence to identify potential anti-cancer uses for existing common drugs. The drugs and cancers studied were; tricyclic antidepressants and both incidence and survival in a number of cancer types, particularly glioma; aspirin and colorectal cancer survival; and angiotensin converting enzyme (ACE) inhibitors and hepatocellular carcinoma (HCC) incidence. A series of studies using The General Practice Research Database as a data source assessed any potential associations: A case-control study for tricyclic antidepressant use and cancer incidence; cohort studies to examine mortality in colorectal cancer and glioma in relation to tricyclic use, and for colorectal cancer mortality in aspirin users; and a case-control study in relation to ACE inhibitor use and HCC. A strong, cancer type specific, dose and time dependant protective effect was found for the incidence of glioma and colorectal cancer. This led to a further study examining mortality for these cancer types in tricyclic users. While no significant protective effects in all-cause mortality of tricyclic users were found, a larger study could still find such an effect in glioma. For aspirin and colorectal cancer mortality, a small but significant reduction in mortality was observed, though these effects were not entirely consistent throughout the study. There were no significant associations found between ACE inhibitors and HCC. These findings contribute to the knowledge of the anti-cancer effectiveness of these drugs, and may assist in designing future clinical studies.

Nitrogen-containing bisphosphonates (N-BPs) are the standard treatment in cancerassociated bone disease due to their ability to inhibit osteoclast-mediated resorption. However, beyond their anti-resorpitive activity there is increasing evidence from preclinical studies to demonstrate that N-BPs can also have an anti-tumour effect, although the mechanism is still unclear. In vitro studies suggest that anti-tumour effects may be due to direct effects on tumour cells (by inhibiting protein prenylation) or via indirect effects on other cell types. The studies described in this thesis sought to answer this question by developing novel approaches and molecular tools. N-BP resistant tumour cell lines were created by over-expressing FPP synthase (the molecular target of N- BPs) by lentiviral transduction of B16 melanoma cells. Although these cells were only modestly resistant to N-BP, such an approach might be a novel strategy for determining whether NBPs directly affect tumour cells in vivo. However, further studies carried out with mice bearing 4T1 mammary fat pad tumours demonstrated that macrophages and myeloid derived suppressor cells (MDSC) internalised a fluorescently-labelled N-BP. Since macrophages and MDSC are important for tumour growth and metastasis, these studies indicate that these myeloid cells may actually be responsible for the anti-tumour effects of N-BPs. The functional effects of N- BPs on MDSC and macrophages remain to be determined. However, an in vitro prenylation assay was developed as a novel and sensitive tool to study subtle changes in protein prenylation and should prove vital in future studies to determine conclusively whether N-BPs inhibit protein prenylation in these myeloid cell types in vivo. Finally, proteomic techniques were used to examine the levels of small GTPases in MDA-MB-231 breast cancer cells and showed that Rab7 appears to be elevated in a clone that preferentially metastasises to the skeleton. This highlights the need for further investigation of the role of Rab7 in skeletal metastasis and as a potential new therapeutic target.

The androgen receptor (AR) is the central component in regulation of the androgen signalling within the prostate gland. Deregulation of the AR activity is frequently involved in the development of prostate cancer. Treatment of advanced prostate cancer often involves chemotherapy and most of these drugs exert their function by generating genotoxic stress such as DNA damage. Although many cellular responses to DNA damage have been clarified over the past years, the effects of genotoxic stress on AR function remain to be elucidated. Here, the effects of genotoxic agents used in chemotherapeutic regimes were investigated in relation to endogenously expressed AR function in the hormone responsive prostate cancer cell line LNCaP. This led to the novel finding that the topoisomerase 11 inhibitors, etoposide and doxorubicin, and the DNA crosslinking agent, cisplatin, inhibited the AR activity. It was further discovered that this loss of AR activity could not be explained by changes in cell cycle distribution, altered nuclear translocation of the AR, reduced expression of the receptor or by induction of apoptosis. Activation of the tumour suppressor p53 is a central component in various cellular responses to genotoxic stress, however, the inhibition of AR activity in response to genotoxic stress was found to be mediated by a mechanism independent of p53 function. Etoposide reduced AR ligand binding within the first hour of androgen exposure, a response only observed to a minor degree after cisplatin treatment. In contrast, cisplatin caused a loss of serine 81 phosphorylation on the AR after 8 hours of drug exposure, which was a response not seen in etoposide treated cells. Interestingly, further studies revealed that at early timepoints both agents inhibited the hormone stimulated recruitment of AR to androgen response elements (AREs) in the promoter and enhancer regions of an AR regulated gene. A possible involvement of MAPK, P13K or cell cycle checkpoint signalling pathways was investigated, but none were found to be directly involved, however, preliminary studies suggest that fully functional HSP90 may be involved in this aspect of AR regulation.

Nogalamycin is a member of the anthracycline family of antitumour antibiotics. These are potent cytotoxic agents and are routinely used in cancer chemotherapy. Though nogalamycin is clinically insignificant, it does exhibit three distinct types of non-covalent binding to DNA. Since most other anthracyclines bind to DNA by only one or two of these mechanisms, nogalamycin is an excellent model with which to probe the interaction of this class of anti-tumour agents with DNA. Here, we investigate the binding orientation and stoichiometry of nogalamycin in adjacent TpG(CpA) (and CpG(CpG)) intercalation sites using a combination of NMR techniques and NOE-restrained molecular dynamics simulations. These methods are also employed to investigate the structure of GNA hairpin loops, which are considered to have important biological functions, and assess how their structure and stability are influenced by the introduction of nogalamycin at an adjacent site. The effect of nogalamycin on extrahelical thymine bases incorporated onto either face of the intercalation sites is also investigated in this context. Binding of quadruplex-specific antibodies to telomeric DNA in Stylonychia lemnae macronuclei has recently been detected using immunofluorescence, providing direct evidence for the formation of quadruplex DNA structures in vivo. Guanine-rich quadruplex structures have been extensively studied by NMR and x-ray crystallographic methods. Previous structural studies have failed to unambiguously resolve the conformation preferred by less-stable A-tetrads incorporated into DNA quadruplexes. Additionally, little effort has been made to address the exact number of ions bound to these adenine-containing structures. This forms the basis of our study into quadruplex DNA. Finally, we endeavour to investigate the extent of hydration of both duplex and quadruplex structures using rMD methods, and to comapre hydration patterns in the liquid- and solid-state.

CD55 is one of the complement regulatory/inhibitory proteins and is over-expressed on a wide range of solid tumours. CD55 is also known to be deposited within tumour stroma and is secreted in an active soluble form, mediated by matrix metalloproteinase-7. The complement cascade forms part of the innate immune system and culminates in cell lysis of targeted cells. As a complement regulatory protein, the primary function of CD55 is to accelerate the decay of complement components preventing formation of the membrane attack complex. CD55 is also known to be a ligand for the T cell early activation antigen CD97, and their interaction has been shown to inhibit the proliferation of activated T cells. This project aimed to develop anti-tumour immunotherapeutics aimed at exploiting CD55 as a tumour associated antigen. Initial strategies were to develop monoclonal antibodies, specific to identified epitopes from within the CD55 protein sequence, capable of binding, and neutralising CD55s decay accelerating activity. Developed antibodies would also have the potential to induce antibody dependent cell cytotoxicity, thus blocking CD55 protection of tumours and mediating an active anti-tumour response. Antibodies were raised specific to CD55 derived linear peptides, which have been used for the assessment of CD55 expression in breast tumour sections. Monoclonal antibodies failed to recognise natively expressed protein on viable tumour cells and alternate strategies were developed. An effective immunotherapy for the treatment of cancer would engage both cellular and humoral mediated responses for effective clearance of target cells. In order to achieve this, a DNA vaccine incorporating a human IgG Fc tail was developed expressing the active sites of CD55, containing HLA-A*201 restricted heteroclitic epitopes. The vaccines were used to immunise HLA-A*201 HHDII transgenic mice and CD55 specific responses were assessed. One of the vaccines analysed, elicited CD55 specific antibodies capable of recognising tumour cells in vitro and also generated epitope specific CD8+ T cell mediated lysis of epitope bearing cells. The frequency of CD55 specific T cells was obtained via antigen specific IFN gamma release ELISPOT assays and the cytokine profile of responses generated was assessed via luminex analysis. In conclusion, CD55 remains a viable target for immunotherapies aimed at CD55 bearing tumours. DNA vaccines encoding modified epitopes are capable or raising cellular and humoral responses to this antigen and further studies should be completed in order to determine anti-cancer effects in tumour bearing models.

Introduction: Cancer of the same origin show considerable heterogeneity in sensitivity to chemotherapy both clinically and in vitro, and show rapid adaptation to chemotherapy based on gene expression. This study tested the hypothesis that anticancer drug exposure could render tumour-derived cells more susceptible to second agents, particularly those with specific molecular targets in survival pathways and with the knowledge of cellular pathways, determine new more effective molecularly designed regimens. Materials and Methods: Single agent, combinational and sequential chemosensitivity of a series of cell lines and tumours was assessed using the ATP-based tumour chemosensitivity assay (ATP-TCA). Sensitivity data was correlated with gene expression, measured by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) in a TaqMan Array following extraction of mRNA from cell samples and standardisation to the housekeeping gene (PBGD). Mutation analysis kits utilising Amplification Refractory Mutation System (ARMS) and scorpion technology were used to establish the presence of activating mutations in EGFR, KRAS, BRAF and PI3K. Results: Heterogeneity in cellular sensitivity to cytotoxic and targeted agents was observed. While gene expression was seen to show some correlation with sensitivity to signalling pathway combination targets, the complexity associated with cellular adaptation prevented the prediction of response to second agent sequential treatment. Discussion: This study has identified potential novel combinations for use in ovarian cancer. This combination of EGFR and PI3K inhibitors has shown greater sensitivity in cell based assays compared with single agent activity and could become the focus of future clinical trials. Successful application of 384 well ATP-based chemosensitivity assays has shown to be a valuable tool for future cell based research. The quantity of viable tumour derived cellular material is diminishing; therefore, methods developed here will continue to provide the means to complete these types of studies in the future.

Folate uses the natural endocytosis pathway via the folate receptor (FR) to enter the cells. Folate conjugation to small or macromolecular therapeutics has hence been exploited for intracellular delivery to, particularly, cancerous cells. This work reports on the expression and functionality of FR in polarised cell monolayer models of respiratory and gastrointestinal mucosa with the view to assess its potential for delivery of folate-modified macromolecular therapeutics either intracellularly or across the epithelium. Four cell lines representing bronchial and intestinal epithelium; cancer-derived intestinal Caco-2 and bronchial cell line Calu-3, and noncancerous intestinal and bronchial cell lines IEC-6 and HBEC were cultured on permeable membranes to produce polarised monolayers. Expression of FR was confirmed by RT-PCR and Western blot analysis for all the tested cell types and shown to be dependent on culturing time. The functionality of the receptor for endocytosis was demonstrated by a model macromolecular folate conjugate (fluorescent ovalbumin-folate (OVA-FA)), whereby significantly higher cellular uptake of the folate-conjugate, relative to non-folate control, was clearly demonstrated. Importantly the data showed that the expressed folate receptor was capable of mediating transport of the macromolecular folate conjugate across (transcytosis) the cells in the polarised monolayers. Preliminary studies led to investigation of the folate mediated uptake and transport of folate modified nanoparticles (NPs). It was shown that folate modified NPs traversed the Calu-3 layers and studies characterizing this transport indicated folate involvement in this process. Adsorption of OVA-FA on the surface of NPs was seen to promote their cellular uptake and transport across the cell layers. To examine the mechanism of cellular uptake and transport of folate modified nanoparticles, various endocytic inhibitors were employed. The study demonstrated an involvement of the caveolar pathway in internalization of folate modified nanoparticles; as judged from a significant reduction of internalization in filipin (inhibitor of caveolar pathway) treated cells. Moreover, the work also showed evidence of transport of folate-modified nanoparticles via the caveolar pathway, since translocation of nanoparticles across the cell monolayer was absent when this path was inhibited. Disruption of actin filament and microtubules caused no difference in cellular uptake of NPs but increased the transcytosis of folate modified NPs. Confocal microscopy, Transmission Electron Microscopy (TEM), Total Internal Reflection Microscopy (TIRM) and Total Internal Reflection Florescence microscopy (TIRFM) were used to confirm and visualize quantitative data. This study also investigated the effects of surface ligand distribution pattern (ligand clustering and density) on the internalization of nanoparticles by Calu-3 cells cultured as polarised layers. The density of the displayed ligand was manipulated by controlling the conjugation level of folate-ovalbumin, while ligand clustering was achieved by co-adsorption of varying mixtures of folate-ovalbumin conjugate (at different ligand density levels) and unconjugated ovalbumin. Increasing ligand density on the nanoparticle surface resulted in increased internalization of modified nanoparticles by the cells, up to a saturation level. Surface ligand density also affected the cellular uptake pathway; from predominantly clathrin to predominantly caveolae-mediated as the ligand density was increased. It was further demonstrated that surface clustering of the folate ligand enhanced cellular internalization of nanoparticles, relative to its dispersed surface distribution.

The chemistry and structure of Poly (glycerol adipate) facilitate its substitution with various pendant functional groups leading to modifications of the physicochemical properties of the polymer. Modified backbones then can be selected based upon the properties of the compound to be incorporated. Thus, this could be explored as a drug delivery system without many of the limitations of commercially available polymers. The aim of this study was investigate whether various polymers and drugs interact in a specific manner and whether the nature of these interactions influence the physicochemical characteristics of the particles and their drug loading and release profile. By investigating drugs belonging to various classes and with different properties it has been possible to correlate properties associated with drugs and pendant functional groups of the polymer which are ultimately responsible for the drug loading and release characteristics. For some drug polymer formulations, good loading and controlled release rates have been achieved. Compared to various conventional polymer systems reported for nanoparticle formulations, poly (glycerol adipate) polymers have also demonstrated the ability to control rate of release of highly water soluble drugs, even from the most hydrophilic polymer backbone in its unsubstituted form. From the various drug loading and release profiles it has been demonstrated that, unlike reported literature, particle size is not the primary factor influencing drug release over the relatively small range of particle sizes seen in this study. Neither is the water solubility of either the drug or the polymer alone responsible for the rapid and uncontrolled release profile from nanoparticles. Thus, Drug polymer interactions are more likely to influence drug loading and release and unlike common reports in the literature, hydrophilicity, molecular weight or concentration of polymer / drug are less likely to affect these parameters in isolation.

The research work presented in this thesis deals with the anticancer activity of four medicinal plants: 'Caralluma tuberculata' (Asclepiadaceae), 'Fagonia indica' (Zygophyllaceae), 'Solanum surattense' (Solanaceae) and 'Arisaema utile' (Araceae) that originate from the North West and Himalayan regions of Pakistan. Through a bioactivity-guided fractionation approach, the crude and resultant organic fractions were tested on cultured breast cancer cells (MCF-7 and MDA MB-468) and colorectal carcinoma cells (Caco-2) in vitro. Five new compounds out of seven in total were isolated from potent fractions of the new medicinal plants using repeated flash column chromatography. Structural elucidation was carried out through a series of spectroscopic experiments (1-D and 2-D NMR, GC-MS, LC-MS). SIngle crystal X-ray structure was determined using X-ray crystallography for the crystalline compounds, which showed a defraction pattern. The apprent IC[sub]50 for compounds (1-6) were estimated from serial dilutions of eight concentrations (0.78-100 [mu]M) of each compound, tested against breast and colon cancer cell lines, using two cell viability assays (MTT and neutral red uptake assays) for 24 h and 48 h treatments. Two new steroidal glycosides, acylated pregnane (1) and acylated androstane (2) glycosides, isolated from the ethyl acetate fraction of 'Caralluma tuberculata' showed highly significant (P<0.001) percentage growth inhibition in Caco-2 cells (IC[sub]50) 1.56-6.25 [mu]M) and MCF-7 cells (IC[sub]50 6.25 - 25 [mu]M), however, oestrogen independent cancer cells (MDA MB-468) were less responsive with IC[sub]50 25 - 50 [mu]M. These steroidal glycosides induced apoptosis in cancer cells as measures of cytoxic activity (NRU, PARP clevage, DNA ladder) on MCF-7, MDA MB-468 and Caco-2 cells were inhibited by pre-treatment with the pan-caspase inhibitor (Z-VAD-FMK). Another pregnane glycoside (3), isolated for the first time from 'Fagonia indica', was found to be more potent in suppressing cell growth (IC[50] 6.25-25 [mu]M), in oestrogen negative breast cancer cells (MDA MB-468,) as compared to oestrogen positive cancer cells (MCF-7). Although a cleaved PARP (89kDa) was detected by Western blotting, cytomorphological alterations and in cells pre-treated with a pan-caspase inhibitor (NRU assay), indicated that the necrosis mode of cell death is more likely. Moreover, three esters: hexadecanoic acid ethyl ester (4), phtalic acid 1-(1, 1-dimethyl-pentyl) ester 2-(2-ethyl-dec-5-enyl) ester (5) from chloroform fraction of 'Solanum surattense', and 5-Oxo-19-propyl-docosanoic acd methyl ester (6) from 'Arisaema utile', showed a highly significant )p<0.001) decrease in cell numbers for MDA MB-468 and Caco-2 cells with apparent IC[50] 6.25-12.5 [mu]M in cell viability assays (MTT and NRU) after 48 h treatment, while MCF-7 cells were less responsive (IC[sub]50 25 [mu]M). Compunds 5 and 6 (first report from a natural source) did not restrict the growth inhibition in MCF-7 and Caco-2 cells, pre-treated with Z-VAD-FMK, which indicated less involvement of Capase-dependent apoptosis, while DAPI staining and Western blots (cleaved-PARP) showed characteristics of apoptosis that suggested the possibility of aponecrosis phenomenon of cell death. In preliminary screening (Western blot and DNA ladder assays), compounds (1-6) were not toxic to normal human cells (HUVEC and U937) and indicated some selctivly between malignant and normal cells.

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