Advanced breast carcinoma is a main cause of mortality affecting women. It is associated with poor prognosis; therefore, understanding the molecular mechanisms of invasive malignancies is critical in order to discover new therapies and to optimise current therapies. Degradation of the Extracellular Matrix (ECM) is a crucial step in tumour growth and invasion, allowing the cancerous cells to metastasize to distant organs and form secondary tumours. Matrix metalloproteinases (MMP) are a family of zinc-dependant endopeptidases that have the capability to degrade the ECM, enabling tumours cells to invade and migrate. Hence, MMP overexpression and/or enhanced activity are positively associated with breast cancer metastasis and invasion. Two MMPs have been shown to be involved in breast cancers, gelatinase A (MMP-2) and gelatinase B (MMP-9). The activity of MMPs is tightly regulated by endogenous inhibitors that are called tissue inhibitors of metalloproteinases (TIMPs). There are four different endogenous negative regulator proteins of MMPs, with TIMP-1 and TIMP-2 inhibiting MMP-9 and -2, respectively. Due to their potential role in tumour metastasis, a great interest has grown in developing novel methods to inhibit the MMPs as there is a direct relation between the expression of MMPs and the invasiveness of the tumours. The Farnesoid X Receptor (FXR) is a nuclear receptor that is highly expressed in breast cancers, and has been reported to be involved in in regulation of MMP and TIMP activity in hepatic and vascular tissues. The rationale of the current study was to investigate whether FXR is a novel regulator of matrix metalloprotease-2 and -9 in metastatic breast cancer cells, and hence may represent a novel therapeutic target. Two FXR agonists were used to measure their effects on breast cancer cells MDA-MB-231, MDA-MB-468 (triple negative), MCF-7 (Estrogen receptor positive) and normal cells MCF10A: chenodeoxycholic acid (CDCA) is an endogenous (low-affinity, low-selectivity) ligand, while 3-[2-[2-Chloro-4-[[3-(2, 6-dichlorophenyl)-5-(1-methylethyl)-4isoxazolyl]methoxy]phenyl]ethenyl]benzoic acid (GW4064) is a synthetic (high affinity, high-selectivity) ligand. Cell viability, protein and mRNA levels of matrix metalloprotease -2 and -9 and TIMP-2 and -1, and cell migration were assessed using both molecular and cellular techniques. Both FXR agonists decreased breast cancer and normal breast cell viability, with the effects more significant in the triple negative cells, suggesting a potential targeting towards aggressive, hard-to-treat cancer cell-types. However, the FXR ligands didn’t alter mRNA and protein levels of MMP-2, MMP-9, TIMP-1 and TIMP-2 intracellularlly or extracellularlly, suggesting that this cytotoxic effect may not be via MMP. FXR ligands also had no effect on breast cancer cell migration, which is consistent with the suggestion that FXR activation has a general cytotoxic effect on tumour cells, but does not directly impact on tumour migration. In conclusion, FXR activation does not impact on markers of breast cancer metastasis, suggesting that its potential as a therapeutic target to prevent tumour progression may be limited. However, the cytotoxic effect on cancer cells is promising, suggesting that these agonists may still possess some potential for breast cancer therapy.

Diabetes and cancer are major health problems because of their high incidences worldwide. A growing body of epidemiological evidence indicats a molecular link between diabetes and breast cancer. Patients with diabetes mellitus have an increased likelihood of developing various types of cancers including breast cancer through the formation of advanced glycation endproducts (AGEs) which lead to cellular and bio-molecular dysfunction. However, the effects of AGEs have been poorly investigated on breast cancer cells. This current study examined for the first time the biological effects of various concentrations of BSA-derived AGEs on the invasive and hormone–independent breast cancer cell line MDA-MB-231 and on non- invasive hormone-dependent human breast cancer cell line MCF-7. Bovine serum albumin was glycated-using methylglyoxal for three days. Crosslinked AGEs were assessed using sodium dodecyl sulphate polyacrylamide gel electrophoresis followed by Coomassie blue staining. Different assays including cell proliferation, migration and invasion through the MatrigelTM with assessment of matrix metalloproteinase (MMP) activity by using Zymogaraphy were performed to investigate the effect of different concentrations of BSA-AGE on both cell lines in vitro. Furthermore, signalling pathways were investigated by Western blotting and using kinexusTM phspho-protein microarray. The expression of the main receptor for AGEs (RAGE) involved on the MDA-MB231 and MCF-7 breast cancer cell lines was assessed by Western blotting and Calibur Flow Cytometer System. The results of this study demonstrated that BSA-AGEs increased MDA-MB-cell, proliferation, migration and invasion through the MatrigelTM associated with an enhancement of matrix metalloproteinase (MMP)-9 activities, in a dose-dependent manner, up-regulated the expression of the receptor for AGEs (RAGE) and of the key signalling protein, phospho-extracellular-signal regulated kinase (p-ERK)-1/2. In addition, the blockade of BSA-AGE/RAGE interactions using anti-neutralizing RAGE antibody reduced the expression of p-ERK1/2. Furthermore, in BSA-AGE-treated cells, phospho-protein micro-array analysis revealed the main enhancement of the over-phosphorylation of (ERK1/2), (p70S6K1), (STAT)-3 and (MAPK) p38, involved in cell survival, cell growth cell cycle and protein synthesis. In contrast, MCF-7 showed stimulatory effects of BSA-AGEs, on cell proliferation and migration, as compared to non-modified BSA. However, BSA-AGEs did not change the weak invasive capacity of MCF-7 cells to cross a reconstituted basement membrane. In addition, BSA-AGEs induced over-phosphorylation of RAGE in MCF-7 cells. The investigation of signalling pathways suggests that BSA-AGEs might contribute to breast cancer development through activation of MAPK pathway and activation of CREB1 transcription factor in MCF-7 cells. Throughout the study, the non-modified BSA had a negligible effect. In conclusion, BSA-AGEs might contribute to breast cancer development and progression of breast cancer. In addition, the up-regulation of RAGE and key phosphor-protein signalling expression induced by BSA-AGEs might be a promising target for therapy to prevent the development of breast cancer in diabetic patients.

Tumour formation may result from the activation of dominant oncogenes or by inactivation of recessive, tumour suppressor genes. The role of such mutations in the development of pituitary tumours has been studied. Tumours from 88 patients, representing the 4 major classes of pituitary adenoma, were investigated. In DNA extracted from matched leucocyte and tumour samples allelic deletions were sought with 15 probes identifying restriction fragment length polymorphisms on chromosomes 1, 5, 10, 11, 13, 17, 20 and 22. Evidence of amplification or re-arrangement of 10 recognised cellular oncogenes (NRAS, MYCL1, MYCN, MYC, HRAS, BCL1, HSTF1, SEA, KRAS2 and FOS) was sought in tumour DNA. Activating dominant mutations of the oncogene Gsalpha were detected using the polymerase chain reaction to amplify exons 7-10 and hybridising the product to normal and mutant allele specific oligonucleotides. Allelic deletions on chromosome 11 were identified in 16 tumours (18%) representing all 4 major sub-types. Deletions on other autosomes were observed in less than 6% of tumours. Three adenomas had deletions on multiple autosomes, two of these were aggressive and recurrent. Mutations of Gsalpha were confirmed to be specific to somatotrophinomas, being identified in 36% of such tumours in this series. No evidence of amplification or rearrangement of other recognised cellular oncogenes was found. Inactivation of a recessive oncogene on chromosome 11 is an important and possibly early event in the development of the 4 major types of pituitary adenoma whilst activating mutations of Gsalpha are confirmed to be specific to somatotrophinomas. Two aggressive tumours were found to have multiple autosomal losses, suggesting a multi-step progression in the development of tumours of this phenotype.

3ʹ,4ʹ,5ʹ-Trimethoxyflavonol (TMFol), a synthetic analogue of the naturally occurring flavonols quercetin and fisetin, has demonstrated putative anti-cancer activity in the prostate. The mechanisms of action that are engaged are largely unknown. Therefore, the work presented in this thesis investigated the mechanisms used by TMFol to compromise cell proliferation in prostate cell lines 22Rv1, PC-3 and PNT2. Furthermore, it investigated the effect of TMFol on prostate cancer development and progression in two separate transgenic models, PBCre4p53floxRbflox and PBCre4Ptenflox mice. Apoptosis and cell cycle distribution were analysed by flow cytometry, while biochemical assays, Western blots, histological evaluations or qPCR were used to analyse senescence, changes in prostate metabolism and the effect of TMFol on mice. TMFol inhibited cell proliferation in vitro without inducing apoptosis. However the reduction in tumour growth in 22Rv1 xenografts, that were fed a 0.2 % TMFol diet, was associated with a significant increase in cleaved caspase-3 staining, an indicator of apoptosis. There was S phase arrest in the PC-3 and PNT2 cells, while there was an increase in senescence-associated β-galactosidase activity in both 22Rv1 and PC-3 cells. TMFol also caused a significant increase in the protein expression of p21 and p53 in 22Rv1 cells. TMFol significantly inhibited the expression of acetyl-CoA carboxylase at the protein and mRNA levels and the expression and activity of mitochondrial aconitase, which could result in the inhibition of fatty acid synthesis and citrate oxidation, respectively. PBCre4p53floxRbflox and PBCre4Ptenflox mice fed a 0.2 % TMFol diet did not display any evidence of tumours in the prostate, but there was no significant difference in the PIN development between the mice on the control diet and those on the TMFol diet. Taken together, TMFol has the potential to induce cell cycle arrest, apoptosis and senescence and to inhibit fatty acid synthesis and citrate oxidation in prostate cancer cells.

The experimental evidence for an immune response to cancer has been reviewed, and the effects of cytotoxic chemotherapy shown to affect nearly all of its aspects. Augmentation of the immune response (immunotherapy) has been discussed in the context of several important agents. It is argued that in advanced cancers the tumour burden is too great for significant benefit from immunotherapy. However in patients with 'minimal residual disease' there is little evidence for impairment of immune responses. When such patients receive adjuvant chemotherapy the number of residual cancer cells is further reduced, but the patient's immunological ability to complete their elimination is seriuosly impaired by the treatment. This situation may be a most suitable opportunity to achieve benefit from immunotherapy, and this study concerns attempts to identify means of achieving this. The work is concentrated on one major arm of the immune response, both in normal rats and some in whom breast cancers were induced. T lymphocyte function was measured in rats by an in vivo (DTH response) and an in vitro (PHA blastogenesis) method. Clear depression was seen following one injection of cyclophosphamide or 5 fluorouracil (5FU) in both normal and tumour bearing animals, and this lasted for at least one month (PHA). The rebound overshoot phenomenon was observed in both groups following 5FU but not cyclophosphamide. Levamisole did not improve the depression of in vitro T cell function produced by cyclophosphamide, but alleviated that following 5FU if administerd after a delay of 3 days. This effect was somewhat marginal but seen consistently in both normal and tumour bearing animals. The combination of glucan with either cytotoxic agent significantly worsened in vitro T cell function, even if the timing of each drug was varied. This observation is interpreted as a directly depressive effect of glucan on T cell function, revealed only in conjunction with cytotoxic therapy. A similar effect was also seen following C parvum but not thiabendazole. The use of a small priming dose of either chemotherapeutic agent did not alleviate the immunosuppressive effect of a subsequently administered large dose. Wide variation of the priming delay for cyclophosphamide did not alter this conclusion. Similarly no benefit was gained either from the regular administration of cimetidine, or the timing of cytotoxic injections to opposing extremes of diurnal rhythms. The difficulties encountered in this field of research and questions for future study are discussed.

The role of an immune response in the development and regulation of tumour growth is outlined. The historical development of immunotherapy in the treatment of human cancer is reviewed and the mechanisms by which immunity may be manipulated in vitro and in vivo with respect to improving cancer immunotherapy are discussed. The experimental studies and laboratory methods finally used are presented and validated in chapter 3. Non-major histocompatibility-restricted cellular cytotoxicity was assessed in a standard 4-hour 51chromium-release assay and peripheral blood cell populations were examined using fluoresceine-conjugated monoclonal antibodies and flow cytometry. Initially in vitro studies of cellular cytotoxicity against cultured tumour cells were undertaken using peripheral blood mononuclear cells (PBMC) obtained from healthy volunteers. Extremely variable levels of cellular cytotoxicity were documented in these healthy donors which were unrelated to age or sex but were positively correlated with numbers of circulating natural killer (NK) cells. The effect on cytotoxicity, of preincubating PBMC with monoclonal antibodies (mAb) and interleukin 2 (IL-2) was studied. Various mAb including those to the CD3 antigen were found to enhance cellular cytotoxicity in vitro. The mechanism of enhancement remained unproven. However, preliminary studies suggested that mAb redirected effector cells by interacting with Fc receptors on the surface of tumour cells. Combinations of mAb had a partially additive effect on enhancement of cytotoxicity. IL-2 also enhanced cytotoxicity in a dose dependent manner although synergy between IL-2 and anti-CD3 was not demonstrated. PBMC were then studied from patients with advanced gastrointestinal tract cancer. Levels of cellular cytotoxicity were not significantly related to tumour extent and were similar to cytotoxicity in healthy donors. Cytotoxicity was mediated mainly by NK cells although lymphocytes coexpressing the CD3 antigen and NK surface antigens were significantly increased in cancer patients compared with healthy donors and also in patients with liver metastases compared to those without liver involvement. In cancer patients with liver metastases, lymphocytes coexpressing the CD3 antigen and NK antigens may play a more significant role in K562 cytotoxicity. Cytotoxicity was also enhanced in vitro by anti-CD3 mAb and I1-2. A clinical study was undertaken to investigate the safety and the immunomodulating properties of administering between 50 mug and 0.5 mug of OKT3 or normal saline to patients with advanced cancer. Patients experienced minimal and self- limiting dose-related side effects. Only one patient developed evidence of cytokine release following OKT3 when assessed by enzyme-linked immunosorbent assays. Depletion of circulating T cells and NK cells occurred following OKT3 which was proportional to the dose administered. Lymphocyte activation assessed by interleukin 2 receptor expression was not detected. Considerable individual variation in cytotoxicity was related to variation in NK cell numbers in blood in both OKT3 and normal saline treated patients. 50 mug OKT3 was associated with a rapid decline in both circulating NK cells and cytotoxicity at 4 hours compared with lower doses of OKT3. The lower doses of OKT3 had a variable effect on cytotoxicity at 24 hours. 20 mug and 5 mug OKT3 was associated with a significant rise in cellular cytotoxicity at 24 hours compared with untreated controls and the 10 mug dose. These results demonstrate that anti-CD3 mAb enhance cellular cytotoxicity in vitro. The effect of OKT3 on lymphocyte function in vivo is dose-related and variable. Low doses of OKT3 may either enhance or depress cellular immunity depending upon the precise dose and time of study following administration. The relationship between the in vitro effects of anti-CD3 mAb on cellular cytotoxicity and their effects in vivo are uncertain. Although low doses of OKT3 may be safely administered to cancer patients further studies are needed to define its potential in human cancer immunotherapy.

Three cross-sectional surveys of acceptance of faecal occult blood testing for colorectal cancer screening, one survey of non-acceptors and one randomised controlled trial of an information leaflet were conducted. These were workplace based schemes in the private and public sectors and opportunistic screening using blood donors as a model. Simple educational leaflets explaining the high frequency of colorectal cancer and screening which addressed reasons for non-compliance were investigated. Subjects completed faecal occult blood tests at home and those with positive results underwent colonoscopy. Completion of tests in general practice in those aged 51 to 70 years was 33% (665/2029) in men and 42% (900/2147) in women. In private industry in subjects aged 41 to 65 years, compliance in men was 25% (425/1703) and in women 32% (40/125). In public industry in subjects aged 41 to 65 years compliance was 32% (53/165) in men and 46% (376/820) in women. With opportunistic screening at the blood donor centre compliance in those offered screening aged 51 to 65 years was 66% (75/114) in men and in women 59% (41/70). The health educational leaflets increased awareness of cancer and screening and raised intention to participate in a 100 subjects accompanying patients to hospital clinics. Reasons for non-compliance addressed in the leaflet, were identified from an interview survey of 81 non-compliers in Market Harborough. Common reasons were the unpleasantness of stool collection, lack of appreciation that healthy subjects should participate, fear of further tests and surgery and intercurrent illness. After piloting the leaflet it was tested in a randomised community controlled trial in general practice in subjects aged 61 to 70 years. The leaflet increased compliance in men from 25% (91/360) to 38% (143/381) (X2=12.9, p 0.001), but was ineffective in women (33%, 134/405, vs 34%, 145/425, X2=0.1,ns). Organisers of screening should consider opportunistic approaches and health education leaflets to increase participation. As compliance in this study was lower than in some other programmes, more work is needed to identify other reasons for non-compliance.

Tuberculosis (TB) remains a major public health issue worldwide. Global TB control efforts are significantly limited by the need of lengthy therapeutic regimens for its causative agent - Mycobacterium tuberculosis (Mtb). Mtb drug response largely depends on its growth rate and a physiological state. Non-replicating and slow-growing mycobacteria are generally more tolerant to drugs targeting cell wall biosynthesis. However, the current study shows that, under certain growth non-permissive conditions, cell wall targeting antimicrobials (ethambutol, isoniazid, cerulenin) can promote Mycobacterium bovis BCG survival; the survival-promoting effect was only observed in sealed non-shaking flasks and accompanied by the accumulation of an unknown volatile compound. This phenomenon was mediated by a transcriptional regulator that controls expression of several ATP dependent efflux pumps and was designated as RaaS (for regulator of antibiotic assisted survival). RaaS binding to DNA was found to be regulated by coenzyme A derivatives of fatty acids (oleoyl CoA and stearoyl CoA). Addition of oleic acid (a precursor of oleoyl CoA) completely abolished the survival promoting effect of ethambutol, confirming the biological importance of this regulatory mechanism. Further investigations were focused on one of the RaaS regulon member – rv3489 that encodes a conserved protein with unknown function. Its over-expression in Mycobacterium smegmatis and Mtb resulted in a growth defect in Sauton’s medium, while the gene deletion in Mtb had no effect on the growth in any media tested or during the macrophage infection. In a pilot animal experiment, the Δrv3489 mutant had a growth advantage in murine lungs at early phases of infection, but was attenuated at later stages. Pull down assay results suggested that Rv3489 interacts with GlgE - a maltosyltransferase that interconnects trehalose and glycogen biosynthesis pathways. These studies lead to a couple of clinically relevant conclusions: (i) dysregulation of efflux pumps by interfering DNA binding of RaaS can kill non-replicating bacteria, but (ii) incautious use of efflux pump inhibitors can promote their survival.

Cancer vaccines are a novel method of treating cancer by harnessing the patient's own immune system to recognise and attack malignant cells. Viral vectors can be used to encode tumour antigens to be presented to the immune system . However this field is hindered by immunity to the viral vectors themselves, which are often more immunogenic than tumour antigens. This thesis describes the production of a novel cancer vaccine based on murine leukaemia retrovirus (MLV), where the enveloped vaccine particles express only tumour antigens on the surface with internal viral proteins physically shielded from B cells. This is designed to stimulate B cells specific for the weak tumour antigens by exploiting T cell recognition of strong viral MHC class II epitopes to confirm their activation. Having cloned tumour antigens HER2 and 5T4 into the genome of MLV, the recombinant virus particles were purified and characterised thoroughly to ensure correct expression and presentation of tumour antigens. A vaccination schedule was also optimised, involving a homologous prime-boost strategy. Several prime vaccines were trialled with different results, highlighting the importance of priming CD4+ T cells and avoiding off-target effects. In vivo testing of the vaccine particles in BALB/c mice showed that mice with pre-existing immunity to MLV (those that were "primed" beforehand) had higher antibody responses against the tumour antigen than non-primed mice. This supports the hypothesis that the presence of viral specific T cells will amplify the immune response to the tumour antigen after the subsequent "boost" vaccination, based on the requirement for T cell help for B cell activation and antibody production. This thesis demonstrates that tolerance to endogenous tumour antigens can be overcome by using a self-adjuvanting viral vaccine particle. This method of homologous prime-boosting has important implications for cancer vaccination and immunotherapy.

No description available.