Cancer is one the on the leading causes of mortality in world, causing 8.2 million deaths in 2012. In light of these statistics, the battle against cancer is ongoing. Nucleoside analogues are a major force in cancer chemotherapy. However, one problem accompanying nucleoside-based therapy is drug resistance, due to the abrogation of mechanisms that are crucial to their transformation to their bioactive metabolites (nucleoside phosphates). The ProTide technology was designed to overcome the limitations associated with nucleoside analogues. The technology enables the delivery of the nucleoside monophosphate into the cell by passive diffusion. Work in this thesis details the application of the aryloxyphosphoramidate and phosphorodiamidate pronucleotide approaches on potent anticancer purine and pyrimidine nucleoside analogues. The work presented in this thesis shows that: I. ProTides of 5-fluorouracil- 2’deoxyuridine (FUDR), the deoxyribonucleoside derivative of 5-fluorouracil (5- FU), were able to overcome several important cancer resistance mechanisms, including active transport and nucleoside kinase mediated activation, illustrated by a potent cytotoxic action in different cancer cell lines. Eight potential candidates were synthesised in large-scale and underwent a comprehensive lead selection, identifying NUC3373 for clinical trials, to start in 2015; II. The successful application of ProTide and phosphorodiamidate technologies to 6-thioinosine and 6-thioguanosine did not improve their activity nor did it help in clarifying their mechanism of action. 6-S-Methyl-thioinosine and its ProTides exhibited far greater efficacy compared to 6-thioinosine; III. Application of the ProTide technology on cladribine provided IV proof of the enhanced potencies of 3’-ProTide derivatives over their 5’-counterparts; IV. 2’-deoxy-5-azacytidine (Decitabine) ProTides did not exhibit an improvement in activity compared to the parent nucleoside in different cell models of cancer; V. The bioactivation mechanisms of ProTides using enzymatic assays were successful. Based on these findings, potential avenues to further explore are the cladribine and 6-S-methyl-thioinosine ProTide families, with the hope to identify new clinical candidates.

One in seven approved anticancer drugs in the UK are nucleoside analogues (NA). However, frequent development of resistances and unpredictable toxicity are crucial drawbacks of these compounds. Some of the main resistance mechanisms against NAs include limited cellular permeability and decreased initial phosphorylation of the NAs, thus limiting the concentration of active NAs inside the target cells. The ProTide approach is a pronucleotide technology that successfully overcomes these drawbacks by releasing the monophosphorylated NA into the cell and has led to multiple clinical candidate drugs. This work was focussed on the application of the ProTide approach to novel and known anticancer NAs with the aim of improving their performance and pharmacological properties. Herein, new synthesis and optimisation of selected pyridine and purine NA with modifications in the base and / or in the sugar moieties, along with different synthetic approaches to build the prodrugs are reported. The anticancer activity of the compounds was evaluated via cell viability assays, and the activation of the prodrugs and resistance to enzymatic degradation was proved via enzymatic assays involving Nuclear Magnetic Resonance (NMR) or spectrophotometric methods. Molecular modelling studies were performed in order to understand the interaction of the ProTides and their metabolites with the enzymes. Finally, the family with the best in vitro activity results was enlarged by developing novel ProTide-related nucleoside diamidate prodrugs, which aimed to further improve their bioavailability and stability characteristics.

Prolonged endocrine therapy is the mainstay of treatment for ER+ breast cancer patients. However, resistance develops in many patients which leads to more aggressive disease. Understanding the mechanisms of acquired resistance that emerge as a consequence of prolonged endocrine treatment remains critical. This study aimed to use gene expression profiling to discover induced mechanisms shared by a panel of MCF7-derived acquired resistant cells that underpin endocrine resistant growth. The in vitro panel represents resistance to oestrogen deprivation, tamoxifen or fulvestrant and includes long-term (3year) models to better-mimic clinical endocrine exposure. Affymetrix 1.0ST microarrays detected 572 genes induced in all resistant models versus MCF7. Over-represented ontologies, pathways and functional classification for these genes revealed induction of oxidative phosphorylation (OxPhos) and TCA cycle enzymes in the resistant models, a finding further confirmed by mass spectrometry. Increased oxygen consumption, NADH dehydrogenase and/or cytochrome C oxidase activity was detected in resistant cells, and targeting with OxPhos inhibitors Metformin or Antimycin A confirmed growth-dependency on OxPhos. Western blotting for AMPK (energy sensor) activity and its downstream anabolic targets (ACC, mTOR/P70S6K) showed Metformin reduced fatty acid and protein synthesis in growth-sensitive endocrine resistant cells. In silico analysis inferred clinical relevance since many TCA/OxPhos genes associated with earlier relapse in ER+ and/or tamoxifen treated patients. Monitoring basal glycolysis (extracellular lactate) and growth impact of 2DG or glutamine restriction demonstrated glycolysis and glutaminolysis also contribute to endocrine resistance. The microarrays furthermore revealed that metabolic kinases PCK2, ALDH18A1 and PFKFB2, and components of cell response to Zn were commonly-induced which may additionally help endocrine resistant growth. This study has revealed increased OxPhos arises as a consequence of prolonged endocrine treatment and is a key bioenergetic pathway sustaining resistance. Since resistant growth is Metformin-sensitive, such targeting of this energy pathway (alongside further antihormones or glycolysis/glutaminolysis inhibitors) could help treat resistance.

Renal cell carcinoma (RCC) represents a group of aggressive tumours of the kidney. These tumours have a high propensity for metastasis and are extremely treatment refractive after disease relapse. Therefore, the identification of new therapeutic targets is of great importance. One such potential target for therapy are cancer stem cells (CSCs). CSCs are populations of cells imbued with a stem cell-like phenotype capable of driving tumour formation, metastasis and chemoresistance. As such, reliable methods for the identification of CSC populations and defining targets important to their functionality, in hopes of developing more potent therapeutics is of great importance. Previous work has found CAV1 to play a significant role in the malignant progression of RCC and also in the maintenance of adult stem cell populations. As such, this work aimed to understand if common markers of CSC phenotype in combination with CAV1 can act indicators of poor prognostic outcome in clinically confined RCC. Furthermore, this work sought to identify CSC populations from RCC cell lines using a panel of surface markers common to embryonic, mesenchymal and cancer stem cells. Then, understand if CAV1 is responsible for driving the CSC phenotype in these CSC populations by regulating one of the major characteristics of CSC biology, self-renewal. Co-expression of CD44 and CAV1 in RCC tumours indicated greatly reduced disease free survival in clinically confined RCC. Additionally, CD44/CAV1 was found to be the most significant covariate in predicting disease recurrence. In vitro analysis, using a panel of CSC related markers, was unable of identifying a putative CSC population. However, CAV1 expression in the VHL positive CAKI-1 cell line was important for the maintenance of clonogenicity. Incubation of CAV1 deficient CAKI-1 cells under hypoxic conditions was able to restore lost clonogenicity. Further iv work revealed that CAV1 maintains clonogenicity in CAKI-1 through activation of STAT3 and -catenin. This suggests an important role for CAV1 in the maintenance of clonogenicity through STAT3 activation in VHL competent RCC.

Natural products play a pivotal role in the treatment of cancer; identification of compounds such as taxanes and the vinca alkaloids were seminal landmarks in natural product drug discovery. Jerantinine A (JA), a novel Aspidosperma alkaloid isolated from plant species Tabernaemontana corymbosa, was previously reported to possess cytotoxic activity against vincristine-resistant nasopharyngeal carcinoma cells and is therefore an ideal candidate for biological investigation. Furthermore, Tabernaemontana corymbosa has been placed in the endangered list of threatened species by the International Union for Conservation of Nature (IUCN) thus making it a priority to elucidate the biological activity of this alkaloid. Herein, we report detailed biological evaluation of JA on various human-derived carcinoma cell lines. Our preliminary screens showed that significant inhibition of cell growth and colony formation accompanied time- and dose-dependent induction of apoptosis in human cancer cell lines after treatment with JA. Dose-dependent accumulations of cleaved PARP and caspase 3 further confirmed apoptosis. Profound G2/M cell cycle arrest was observed 24 h after treatment in all cell lines. Characteristics of mitotic arrest including inhibition of tubulin polymerisation, microtubule disruption, and aneuploidy were clearly observed. DNA fragmentation was also evident in cells treated with JA. Indeed, significant increases in phosphorylated-γH2AX were indicative of DNA damage caused by double strand breaks and were relatively similar to levels caused by vincristine. Investigations into JA’s ability to overcome vincristine resistance demonstrated that it is not a substrate of Pgp. The role of reactive oxygen species (ROS) in acquired resistance and cell death have also been widely studied. JA induced significant levels of ROS in treated cells, possibly contributing to their apoptotic destiny. Proteomic analyses also corroborated the phenotype of JA-treated cells with increased expression of ROS-neutralising enzymes, aberrant expression of proteins involved in the spindle assembly checkpoint critical to mitosis, and decreased expression in all tubulin proteins detected by LC-MS/MS. A genome-wide RNAi screen revealed several candidate genes involved in mediating sensitivity to JA. The genes corresponding to c-Jun-N-terminal kinases, JNK1/2, were selected for subsequent investigation based on their involvement in multiple pathways that were identified using bioinformatic tools. JNK1/2 were knocked down in MCF-7 and MDA-468 cells and then treated with JA. MTT assays revealed some loss of sensitivity, suggesting that these proteins were indeed involved in mediating cell sensitivity to JA.

Breast cancer is the second-leading cause of cancer related death in women and future therapies may involve the strategic use of previously developed drugs, which have established toxicity profiles and are often ex-patent. The alcohol-deterrent disulfiram has been proposed as an anti-cancer agent, based on its capacity to interact with copper-dependent processes. However, little has been done to determine the relationship between this drug and zinc, despite knowledge that disulfiram binds this metal and zinc levels are dysregulated in breast cancer. This thesis investigated whether zinc is an important factor when considering disulfiram efficacy as an anti-breast cancer agent. Disulfiram was toxic to MCF-7 and BT474 breast cancer cell lines and produced a striking time-dependent biphasic toxicity response between 5-20 μM. Co-incubation of the drug with low-level zinc removed this effect, suggesting that the availability of extracellular zinc significantly influenced disulfiram efficacy. Structure-activity relationship studies with disulfiram analogues revealed the biphasic effect could be influenced by altering the size of chemical groups bound to the drug’s nitrogen atom. Live-cell confocal microscopy using fluorescent endocytic probes and the zinc dye Fluozin-3, coupled with the development of a complimentary Fluozin-3 flow cytometry assay found that disulfiram rapidly increased zinc levels in breast cancer cells specifically in endo-lysosomes. This indicates that the ii cytotoxic effects of this drug may be due to acute zinc overload. Further investigation into disulfiram effects on lysosomes suggested that the drug disrupts lysosomal membranes and releases hydrolytic enzymes into the cytosol. Lysosomal membrane permeabilisation has been demonstrated to promote apoptosis and may be a mechanism to explain disulfiram cytotoxicity in breast cancer. This could have important clinical implications in situations of high intracellular zinc as seen in breast tumours, indicating that breast cancer cells may be more susceptible to the zinc ionophore action of disulfiram than non-cancer cells.

This study aimed to explore Occupational Therapists’ narratives of “the self” as a therapeutic agent, linking personal development and insight to professional development as an Occupational Therapist (OT). Three female, newly qualified OTs constructed a series of six arts based narratives, using creative media, over the course of three workshops. The narratives developed from an initial exploration of “the self as therapist” then continued through individual exploration of emergent personal themes. Each pictorial narrative was presented verbally to the group and the presentation and discussion of the images were videotaped. The transcribed stories, alongside the artwork, were analysed and revealed clear evidence that personal narratives have the potential to be active, dynamic processes with important implications for therapeutic practice, education and research. The study highlighted the tension generated by conflict or dissonance between the therapist’s sense of self and their professional role. Exploration of this conflict using art and narrative approaches showed how active story telling around challenging issues can lead to greater personal insight, autonomy and resolution through the re-integration of concealed aspects of the self.

The re-emergence of plague as a world-wide health concern and the potential risk posed by bioterrorism has led to an increased interest in available treatments for the disease. The bacterial DNA adenine-N6 methyltransferase, Dam, is involved in the regulation of a range of pathogenic bacteria and has been validated as a target for the development of antimicrobial agents with activity against Yersinia pestis, the causative agent of plague. The lack of a functionally similar enzyme in mammals suggests that highly selective Dam inhibitors could be developed. A coupled, real-time break light Dam activity assay has been optimised for HTS, and assays for the validation and characterisation of screening hits have also been developed. Screening of random and in silico enriched compound libraries, and the subsequent application of counter-screening and hit confirmation assays, resulted in the identification of a single viable lead, (4-(N-(2-hydroxyethyl)sulfamoyl)phenyl) stibonic acid (13776). Screening of compounds analogous to 13776 identified a series of arylstibonic acids with activity against Dam. Kinetic characterisation of the most potent arylstibonic acid, 4-stibonobenzenesulfonic acid (13746), revealed a DNA-competitive mode of action, and a Ki of 6.46 ± 0.07 nM. However, selectivity assays have revealed a potentially non-specific mode of action for the stibonic acids, which have shown activity against a range of DNA and protein binding enzymes. Yersinia cell culture experiments have shown a single compound, (3-((2-hydroxyethyl)carbamoyl)phenyl)stibonic acid (13782), to be capable of penetrating Yersinia cells and partially inhibiting methylation, and mRNA profiling experiments have shown 13782 to induce a statistically significant change in several genes involved in the pathogenicity of Y. pestis. Attempts at resynthesising 13782 have proved challenging, with only a fraction of the activity of the original sample reproduced. HPLC analysis of the original and resynthesised samples has shown the former to comprise two components, with only one present in both samples. The in vitro evaluation of a series of bisubstrate analogues designed to mimic both the methyl donor S-adenosylmethionine (AdoMet), and the methylation target (adenine) has shown that substitution of the AdoMet sulfur for nitrogen results in a significant but not total loss of activity. Furthermore, the addition of a bicyclic heteroaromatic adenine analogue mimic to this scaffold led to an increase in potency and selectivity for Dam over the human cytosine methyltransferase DNMT1 but a reduction in selectivity for Dam over the restriction enzyme DpnI. These results suggest that a selective and potent Dam inhibitor can be obtained by carefully modifying both components of the bisubstrate analogue inhibitor.

Background: Women taking AIs (Aromatase Inhibitors) as treatment for breast cancer commonly experience joint pain and stiffness (aromatase inhibitor associated arthralgia; AIAA) which can lead to early discontinuation of treatment. Exercise is often recommended and there is preliminary evidence it might prove helpful. Nordic Walking is a popular form of exercise in women with breast cancer, and based on a biopsychosocial model, could provide additional benefits over normal walking alone. There is a need to find interventions for this problem; therefore a study was designed to determine the acceptability and safety of a Nordic walking intervention in women with AIAA, and to test the feasibility of a proposed randomised controlled trial in terms of recruitment, methods and measures. Methods: A feasibility study was carried out in a sample of women with AIAA using a randomised control design, with a waiting list control. Forty women were recruited and randomised to either intervention (six weeks of supervised group Nordic walking training followed by six weeks of 4 x 30min/week self managed Nordic walking) or enhanced usual care. Data were collected on feasibility outcomes including recruitment, acceptability (attrition and adherence), safety, and research design issues. Outcome data (pain, depression, quality of life & self-efficacy) were collected at baseline, T1 (following supervised group Nordic walking training) and T2 (following self managed Nordic walking). Findings: The recruitment rate (25%) was comparable to other breast cancer exercise studies, suggesting that there was interest in this type of intervention despite joint pain. Attrition was low (10%) and safety demonstrated. In the intervention group, adherence was high for weekly supervised Nordic walking sessions (>90%) but low for self managed sessions (average of two sessions per week, with most (70%) only managing one), although higher exercise frequencies were attained when all aerobic activity was considered together. Participants in the control group also reported increased physical activity, mainly through normal walking. Most of the outcome measures used appeared suitable for use, demonstrated responsiveness to change and gave support for using a biopsychosocial model of pain. Improvements in pain and other outcomes were demonstrated in both the intervention and control groups, possibly as both increased their physical activity. Conclusions: Our findings indicate that women with AIAA may not adhere to an intensive programme of self-managed NW; however, increasing physical activity is feasible in this population, and may improve symptoms. A future trial should test a physical activity intervention including a supervised component throughout to maximise adherence.

Pulmonary arterial hypertension (PAH) is a progressive and debilitating disease characterised by increases in pulmonary vasoconstriction and excessive remodelling of the pulmonary arteries. Together, these processes lead to sustained elevations in pulmonary arterial pressure, right heart failure and eventual death if left untreated. Despite the number and variety of treatment options available, the survival rate in incident and prevalent cases of PAH remains poor. Therefore, a better understanding of the pathobiology of PAH is required to generate novel therapeutic approaches with improved efficiency in patients. In PAH there is a well described gender bias. Women are consistently reported to represent up to 75% of the total PAH population; however, the reasons for this female predominance remain unclear. Recently, estrogen has been implicated as a major risk factor, for example, elevated estrogen levels and alterations in estrogen metabolism are closely correlated with PAH development in females. The role of testosterone in PAH is currently under investigated. Effects of estrogen are mediated through two classical estrogen receptors (ER)-α and –β, or the novel G-protein-coupled estrogen receptor (GPER). Expression of all of these receptors is identified in pulmonary vasculature, including in smooth muscle and endothelial cells. The role they play in PAH pathogenesis in females is largely undetermined. Given the diverse effects of estrogen described in the pulmonary vasculature during PAH, for example, proliferative effects in pulmonary artery smooth muscle cells (PASMCs), we hypothesised that estrogen receptors play an integral role in PAH in females. To examine this, we used both translational and experimental studies to characterise ERs in PAH. Chronic hypoxic male and female mice, and mice over-expressing the serotonin transporter (SERT+ mice), which demonstrate female susceptibility, were used to investigate the effects of an ERα antagonist in vivo. GPER knockout mice were also investigated in chronic hypoxia. In situ and in vitro studies in human PASMCs with ER agonists and antagonists added clinical relevance to our findings. In addition, testosterone manipulation was investigated in male mice by castration in vivo. Immunohistochemistry, immunoblotting and qRT-PCR analysis demonstrated that ERα was increased in PASMCs and pulmonary arteries from female PAH patients and chronic hypoxic mice, respectively. On the other hand, ERβ was decreased in PAH and hypoxia. It was also observed that females expressed higher levels of ERα in PAH compared to males whereas ERβ was lower in females. PAH was assessed by measuring right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH) and pulmonary vascular remodelling and muscularisation. Chronic hypoxia induced-pulmonary hypertension (PH) was attenuated in female mice dosed with the ERα antagonist MPP, shown by marked reductions in RVSP and pulmonary vascular remodelling. Hypoxic male mice remained unaffected by MPP treatment. Spontaneous PH and chronic hypoxia induced-PH observed in female SERT+ mice were reversed by treatment with MPP. Immunoblotting and qRT-PCR analysis revealed that the possible mechanism involved in the beneficial effect of MPP in females in vivo involved restoring the dysfunctional bone morphogenetic protein receptor-2 (BMPR2) axis observed in PAH. This effect was only observed in female mice. In addition, chronic hypoxia induced- PH in male and female mice was unaffected by GPER deletion. Expression of GPER between female non-PAH controls and PAH patients was unchanged. In isolated human PASMCs estrogen induced proliferation was inhibited by MPP, but not PHTPP or G15, an ERβ and GPER antagonist, respectively. The ERα agonist, PPT stimulated proliferation of human PASMCs. Both estrogen and PPT induced proliferation was dependent on downstream PI3K/Akt and ERK MAPK activity. In males, testosterone deprivation by surgical castration had no effect on chronic-hypoxia induced PH. RVSP, RVH and pulmonary vascular remodelling were unchanged in hypoxic castrated mice relative to sham controls. Testosterone levels, assessed by enzyme linked immunosorbent assay (ELISA) demonstrated no effects of hypoxia on plasma testosterone levels. Testosterone levels were approximately halved by castration. qRT-PCR analysis showed that in mouse lung there were also no difference in expression of the androgen receptor (AR) and 5α-reducatse, the testosterone metabolising enzyme. Testosterone had no effect on proliferation of human PASMCs, although its primary metabolite, dihydrotestosterone (DHT), stimulated proliferation in a dose-dependent manner. In summary of these findings, we have identified an ERα-dependent mechanism of PAH in females, but not in males. ERα is noticeably increased in female human PASMCs from PAH patients compared to male PAH patients. Additionally, ERα activation in female human PASMCs leads to proliferation driven by PI3K/Akt and ERK MAPK activation. Treatment with an ERα antagonist attenuated the development of chronic hypoxia induced-PH in females but not males, and reversed PH in SERT+ female mice. We demonstrate that the mechanism attributed to the beneficial effect of MPP in vivo involved restoration of the dysfunctional BMPR2 signalling axis. Our results suggest that increased ERα expression may drive PAH development in females. Furthermore, we demonstrate that ERα does not play a key role in the development of hypoxia induced-PH in male mice. In addition we conclude that testosterone does not contribute to chronic hypoxic-PH observed in males. We suggest that altered local synthesis and metabolism in the lung and right ventricle may however, facilitate progression of established PAH in males and worsening survival rates. Overall, our results provide evidence for ERα in PAH development and implicate targeting ERs as a novel therapeutic target in PAH treatment.