Colorectal cancer is a major public health problem and a leading cuase of cancer deaths in the western world. At present, three monoclonal antibodies (mAbs) namely anti-epidermal growth factor receptor (EGFR) cetuximab and panitumumab and anti-vascular endothelial growth factor mAb bevacizumab have been approved for the treatment of patients with metastatic colorectal cancer. However, many patients simply do not respond, or the cancer aquires resistance following a short course of therapy. The aim of this study was to develop a new panel of mouse monoclonal antibodies directed against other cell surface antigens which are over-exoressed on human colorectal tumour cells using hybridoma technology and to investigate their potential as diagnostic and therapetic agents. A panel of human colorectal cancer cell lines, established from colorectal cancer patients at different stages of the disease, were employed as the source of immunogen for immunisation of a group of mice. Seven novel mouse hybridomas were developed, secreting seven novel mAbs named KU2.77, KU2.90, KU3.39, KU3.47, KU3.61, KU4.76 and KU4.94. Following purification, the binding efficacy of each purified mAb to a large panel of colorectal tumor cells, human foreskin fibroblast (DE532) and human immortalized keratinocyte (HACAT) cells was determined using ELISA and flow cytometry. All seven mAbs are directed against cell surface antigens which are over-expressed on human colorectal cancer cells. The differential binding of these mAbs to the various human colorectal tumour cells and to the other cells suggests that these antibodies may be directed against at least three distinct over-expressed cell surface antigens. Interestingly, of the seven mAbs examined, KU3.47 and KU4.94 did not bind to DE532 and HACAT cells suggesting that the antigen recognised by these two antibidies may be tumour specific antigens. at 300nM, with the exception of mAb KU3.39 which induced slight growth inhibition of CCL-228 and Colo2 cells, the growth of other colorectal cancer cells were not inhibited by the any of the antibodies while only two mAbs (KU2.77 and KU2.90) inhibited the migration of CCL-228 cells. Out of the seven novel mAbs, KU3.47, KU4.94 and KU3.61 stained their respective antigens in formalin-fixed, paraffin-embedded, colorectal tumour cell pellets, suggesting that these antibodies are directed against a sequential determinant on the antigen and could therefore have potential for use as tools for investigating the expression pattern, prognostic significance and predictive value of such antigens in cancer patients. The preliminary results of immunoprecipitation followed by mass spectroscropy revealed that three newly developed mAbsKU2.77, KU3.39 and KU4.94 may recognise or cross react with integrin alpha-3, Large neutral amino acid transporter small subunit1 (LAT-1) and apo-lipoprotein A-IV respectively. Since all seven novel mAbs are directed against over-expressed cell surface antigens, they could form excellent tools for use in basic research for investigating the role of such antigens in the progression of human cancers. Further investigations are warranted to identify the target antigens recognised by each of the novel antibodies and to determine their full potential as diagnostic agents and therapeutic agents when conjugated to various toxins, drugs or radioisotopes.

No description available.

Most human cancers carry mutations in cell cycle regulators that result in deregulated Cdk activity, which can either be amplification of the cyclins, elimination of the Cdk inhibitors or mutations in the Cdks. These modifications have a high prognostic value. Cdk2 activity has been shown to be upregulated in different kind of tumours (mammary and prostate carcinomas, and lymphomas) due to the mutation of its regulators, p27KiP1 and cyc/in E; and this alteration has a high prognostic value. Moreover, an insensible INK4 point mutation in Cdk4 has been described in human melanomas. To evaluate the importance of Cdks in neoplastic development, the loci encoding Cdk4, Cdk6 and Cdk2 were ablated to study the effect of Cdk deficiency in tumour development. To this end, the corresponding Cdk knock out mice were crossed with the K_Ras+/LSLG12V;RERertert strain that carries an endogenous K-Ras oncogene whose expression is dependent on Cre-mediated recombination. Postnatal expression of this oncogene leads to the development of lung adenomas and adenocarcinomas. Primary MEFs derived from K- Ras+ILSLG12V;RERrrtlert embryos lacking either Cdk4, Cdk6 or Cdk2 displayed decreased proliferation in culture and prevented the growth in low serum condition. However, no obvious differences were detected in immortal MEFs regardless of the missing Cdk.

This project investigated the development of novel anticancer agents with good efficacy and selectivity. Cryptolepine is an alkaloid found in the roots of West African climbing shrub species including Cryptolepis triangu- laris and Cryptolepis sanguinolenta. Cryptolepine is 5-methyl-1oH-indolo [3, 2-b]quinolone, and was first identified as an antimalarial agent, but also acts as an anti-cancer agent by intercalating into DNA and also inhibiting topoi- somerase ll and other key enzymes. Studies elsewhere have shown the mode of action of cryptolepine in vitro appears to be unaffected by drug re- sistance mechanisms identified. In this project a number Cryptolepine ana- logues have been made, modifying key positions in order to enhance DNA binding. The aim of this study was to attach halogens (F, Cl, Br and I) and alkyl amino or amido side chains at the 11-position and then test these mole- cules for anticancer activity. It was anticipated that these nitrogen containing side chains might interact with the sugar-phosphate backbone of DNA to give improved binding and hence interfering with topoisomerase II and related enzymes such as helicase and hence enhancing cytotoxicity. Fluorescence microscopy was used to investigate whether the derivatives reach the cell nucleus. In conclusion, these studies have shown that novel amino- and halogenated cryptolepine analogues have greater in vitro cytotoxicity than the parent compound and are important lead compounds in the development of novel potent and selective indoloquinone anti-neoplastic agents.

Solid dispersions have been employed as a method for improving the dissolution, and hence the bioavailability, and targeting the colon by Class II poorly-water soluble drugs. This project aims to enhance the solubility of the poorly water-soluble drug gefitinib (ZD), which is the first selective epidermal growth factor (EGFR) tyrosine kinase inhibitor that blocks signal pathways implicated in solid tumour growth and metastasis. Furthermore, to target the colon to improve the efficacy and to reduce the adverse effects of ZD by formulation as solid dispersions using spray drying. Methylmethacrylate polymers such as Eudragit S 100 was used for colon targeting, and Polyvinylpyrrolidone (PVP), Hydroxypropyl methyl cellulose (HPMC) were used to enhance the solubility of the drug. A rapid, cost-effective and precise reverse phase HPLC method has been developed to quantify ZD in the formulated solid dispersions. This method was validated using the recommended procedure, and it met the pharmaceutical industry guidelines that are recommended by the ICH and FDA. The developed method of analysis was reproducible, specific, and has demonstrated a isocratic separation mode of ZD. Differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD_, scanning electron microscope (SEM), and attenuated total reflectance (ATR) were used to investigate the crystalline structure changes of ZD in the dispersion as well as the molecular interaction between the drug and polymer, Dissolution studies (DS) were performed across a range of pH values. Formulations were evaluated by cytotoxicity studies (MTT & Neural red tests) using Caco-2 cell line. A rotating modified cylinder method was used to evaluate the duration of adhesion of HPMC and PVP formulated solid dispersions to excised goat intestine. DSC and XRPD studies confirmed that the spray dried dispersions were amorphous. Infrared spectroscopic experiments revealed the formation of hydrogen bonds between the drug and the two polymers used in the study. Dissolution studies showed a carrier controlled drug release that was achieved with solid dispersion containing a 10% drug loading with 80:20 PVP- Eudragit S 100, and HPMC- Eudragit S 100. The morphological differences between the pure drug, physical mixtures and solid dispersions were evident from the SEM images. The images revealed that the pure drug particles were needle shaped; the particle size was reduced in the solid dispersions that subsequently enhanced the drug dissolution profile. The formulated solid dispersions were subjected to accelerated storage conditions (40 [degrees]C [plus or minus] 2 [degrees] C/75%RH [plus or minus] 5%RH) over the period of 6 months. The presence of strong molecular interactions between drug and polymers and the high glass transition temperature of the resultant solid dispersions ensured they remained amorphous for at least six months under accelerated storage conditions. The mucoadhesion study showed that the HPMC solid dispersion formulations had a higher duration of adhesion than PVP solid dispersion formulation. The placebo formulation containing only the polymers showed no toxic effect on Caco-2 cell line in the cell viability assay. Therefore, it can be safely concluded that the formulated solid dispersions have no initial inhibitory effect compared to the pure drug.

Breast cancer is the most common malignancy among females. Although considerable progress in disease detection and treatment has greatly improved survival, this entails the endurance of sequential treatment combinations. Such treatments are associated with cancer related fatigue (CRF), a debilitating form of fatigue that often persists for months or years post treatment, and a reduction in cardiovascular health that increases the risk of cardiovascular disease.

No description available.

Despite recent advances in diagnosis and treatment of breast cancer, it still has the highest incidence and mortality of any cancer among women worldwide, and is responsible for more than half a million deaths every year. In recent decades, increased expression and activation of the human epidermal growth factor receptor (HER) family members has been identified in a wide range of cancers. In particular, HER2 is overexpressed in around 25% of all breast cancers and is often associated with poor patient prognosis. As a result, a number of anti-cancer agents targeting HER2 and other members of the HER-family have been approved for the treatment of breast cancer, including the anti-HER2 monoclonal antibodies trastuzumab and pertuzumab, the reversible dual EGFR/HER2 small molcule tyrosine kinase inhibitor (TK1) lapatinib, the antibody-drug conjugate T-DM1, and, more recently, the irreversible pan-HER TK1 neratinib. Despite these advances, however, many patients with HER2 positive (HER2+) breast cancer do not respond to treatment, and many who do later relapse with acquired resistance to the original treatment. The aim of this study was to investigate the sensitivity of a large panel of breast cancer cell lines to treatment with various types of HER-family TKIs, including reversible EGFR-specific, dual EGFR/HER2, and pan-HER TKIs, and irreversible pan-HER TKIs. Additionally the effect of other TKIs with differing targets, including imatinib (BCR-Abl/c-Kit/PDGFR TKI), dasatinib (BCR-Abl/c-Kit/Src TKI), NVP-AEW541 (IGF-1R TKI), crizotinib (ALK/c-Met TKI), and the chemotherapeutic agents paclitaxel and gemcitabine, on the growth of these breast cancer cell lines was also investigated. The association between receptor tyrosine kinase expression and sensitivity of breast cancer cell lines to these agents was also assessed. Furthermore, the mechanisms of action of these agents and their effect on downstream signalling, cell cycle distribution and cell migration were also examined. Of the HER-family TKIs, the second-generation irreversible pan-HER TKIs, particularly afatinib and neratinib, were more effective at inhibiting growth and migration of breast cancer cell lines compared with the first-generation reversible inhibitors (e.g. erlotinib, lapatinib, sapitinib). Furthermore, the irreversible pan-HER TKIs were more effective than the reversible HER-family TKIs at inhibiting phosphorylation of HER-family members and downstream signalling molecules Akt and MAPK. Of the other TKIs, dasatinib was the most effective at inhibiting growth and migration of breast cancer cells, particularly the triple negative cell line MDA-MB-231. Of the chemotherapeutic agents, paclitaxel was the most consistently effective at inhibiting growth of breast cancer cell lines. Combined treatment of TKIs and chemotherapeutic agents were synergistic in some breast cancer cell lines, but no combination was synergistic in all the cell lines tested. Acquired resistance to both lapatinib and afatinib was accompanied by cross-resistance to all other HER-family TKIs and increased sensitivity to dasatinib and gemcitabine. No significant association was found between the expression levels of HER-family members and response to treatment, with the surprising exception of the irreversible pan-HER TKI canertinib. In addition, the role of human papillomavirus (HPV) in breast cancer was also investigated using breast cancer tissue arrays, and of 100 breast cancer tissue samples examined, 24 were found to be positive for the high-risk HPV oncoprotein E7. However, no association was found between E7 positivity and the expression of EGFR, HER2, oestrogen receptor (ER) or progesterone receptor (PR). Further investigations into the potential of irreversible pan-HER TKIs and the BCR-Abl/c-Kit/Src inhibitor dasatinib,as well as the potential benefits of using HER-family TKIs in combination with dasatinib, NVP-AEW541 and gemcitabine, in the treatment of different subtypes of breast cancer are warranted.

There is abundant evidence supporting the health benefits physical activity in cancer survival. Exercise per se is associated with positive physical and psychosocial benefits for survivors of solid tumours. There are limited available research data on blood borne cancers. Lymphoma is one such haematological cancer where survivors often experience decrements in psychosocial, physical functioning and quality of life (QoL) domains. A minority (~25%) of lymphoma survivors meet the recommended public guidelines for exercise. Further to this, the work of Bellizzi and colleagues (2009) indicates that QoL decrements often persist for years following treatment. Conventional wisdom dictates that exercise is likely to be an effective means of alleviating some adverse outcomes from blood borne cancers but this hypothesis is largely untested to date. Further to this, the theory of planned behaviour (TPB) has been shown to provide effectual model for predicting exercise behaviour amongst cancer survivors but known to differ by tumour type. Therefore, the aims of the present thesis were to determine the effects of 12-weeks of a combined aerobic and resistance training programme (CARP) on QoL and health related outcomes in Hodgkin’s lymphoma (HL) and Non-Hodgkin’s lymphoma (NHL) survivors. The thesis focused specifically on four main aims; Aim 1 the primary aim was to identify whether a 12-week CARP is effective at improving QoL in HL and NHL survivors. Secondary Aims were to; Aim 2 to determine whether a 12-week CARP is effective at improving standard measures of muscle function and cardio-respiratory fitness in HL and NHL survivors. Aim 3 to examine whether a 12-week CARP affects inflammatory environment and/or immune function in HL and NHL survivors. Aim 4 to identify whether theory of planned behaviour (TPB) may be an effectual model to predict exercise intention in HL and NHL survivors. In realising these aims, a parallel group randomised control exercise trial (RCT) was conducted with two components. Forty-one (n=41) HL and NHL survivors completed the trial at St George’s hospital, London. Participants, who had completed chemotherapy or radiation treatment (<6 months), were stratified according to tumour type and randomly assigned to either control (CON; n=21) or intervention (INT; n=20). The intervention consisted of a combination of 12-weeks supervised aerobic and resistance training (CARP) whilst the control group received usual care. The first component consisted of three measurement phases; baseline (To; n=41), post-intervention (T1; n=41) for all measurements, and 12 months follow-up (T2; n=15) for qualitative measures. A representative sample (n=6) from the intervention group took part in a focus group to explore participant perception of the impact of the CARP. QoL was assessed using the previously validated European Organization for Research and Treatment of Cancer Quality of Life (EORTC-QLQ-30) questionnaire. Secondary outcome measures consisted of health-related quality of life (HRQoL) determined by Functional Assessment of Cancer Therapy in Lymphoma (FACT-Lym); Mood disturbance and fatigue were determined using Profile of Mood States (POMS) questionnaire; anxiety and depression were determined using Hospital Anxiety and Depression scale (HADS). Participant cardiorespiratory fitness was assessed using the Balke-ware treadmill test, muscle function assessed by grip strength and muscle endurance tests. Blood was sampled using the standard venepuncture method followed by radioimmunoassay to determine interleukin 6 (IL-6) and c-reactive protein (CRP) concentrations. In order to identify determinants of exercise intention and behaviour in HL and NHL survivors, a second component to the trial utilised a validated TPB questionnaire, assessed at baseline (To; n=41), and post-intervention (T1; n=41). Data were analysed using SPSS version 18.0 using appropriate statistical functions. Statistical significance was set at p<0.05. Data are presented as means i standard deviations (S.D.). Results demonstrate that study adherence between To and T1 was 87.2% (41/47) with a large accession rates at 12 months follow up (15/41). Linear mixed models analysis was used to examine subjectively reported outcomes. Clinically relevant improvements in QoL were achieved in both groups at T1. HRQoL, a domain of QoL, increased with exercise; the improvements were both clinically relevant and statistically significant. Subscales of QoL and HRQoL that significantly improved with exercise are social function (p=0.020), emotional well-being (p=0.029), and functional well-being (p=0.025), as well as functional lymphoma specific concerns (p=0.034). Mood disturbance was unchanged in either group, physical function improved only in the control group (p=0.049). Both groups showed improved (p<0.05) physical well-being, vigour, reduced fatigue, and increase in subjectively reported amount of physical activity (IPAQ) as time passed from the end of treatment. At follow-up, HRQoL, lymphoma concerns, fatigue, and the trial outcome index significantly improved in both groups (p<0.05) from baseline; anxiety significantly increased in the intervention and anxiety, physical well-being, and functional well-being improved in the control group. Both groups reduced physical activity at follow-up. Predicted aerobic capacity showed a trend towards an increase, whereas resting heart rate (p=0.041) abdominal muscle endurance (p=0.018) significantly improved in the [NT group with a concomitant trend for a decrease in the CON group. However, this did not reach a level of significance. Although both groups experienced worsening of pulmonary function post intervention, this only reached a level of significance in the ]NT group. No significant changes in either IL-6 or CRP were observed during the study. ANOVA and MANOVA were used to analyse physical outcomes. Regression analysis was used to determine the predictive value of the TPB variables upon intention to exercise, and TPB variables and intention upon actual behaviour. Simultaneous Multiple Regressions were used first to determine the equation for each model. Stepwise Multiple Regressions were used to examine the impact of each variable on the dependent variable to find the best model of prediction. At baseline (both INT and CON groups collapsed to one) the model predicts intention (68.6%), but prediction of variation in actual behaviour is low (36.2%); self- efficacy (13:0.495) and social support (13:0.469) predict intention to exercise among lymphoma survivors and self-efficacy (B=0.609) alone predicts actual behaviour at To. At T1, the model predicts 77.0% of the variation in intention amongst the CON group but only 14.7% of actual behaviour; attitude (B=0.864) predicted intention to behave. Amongst the exercising group, the model predicts 61.5% of the variation in intention, but only 19.2% of actual behaviour; social support (B=0.800) predicts intention to exercise. None of the determinants significantly predicted actual behaviour at T1. The current thesis presents the first data in examination of the impact of a CARP amongst post- treatment lymphoma survivors. The exercise training intervention significantly improved HRQOL and psychosocial well-being. This is noteworthy as lymphoma survivors are often burdened with reduced HRQOL and psychosocial morbidity. Although predicted aerobic fitness levels were statistically unchanged in INT following the intervention, the trend towards an improvement indicates that either an increase in exercise programme length or intensity of exercise sessions may achieve statistical improvement in future studies. The findings from this thesis indicate CARP to be effective in improving psychosocial outcomes in lymphoma survivors. At 12-month follow-up, reduced physical activity was associated with increased anxiety; functional and physical well-being did not improve despite increases seen in CON.

Despite many advances in cancer diagnosis and therapy in the last few decades, pancreatic cancer remains one of the most fatal types of human malignancies, with a 5-year survival rate of less than 6%. One of the major contributing factors for the poor prognosis of this malignancy is the lack of specific markers for its early detection. In addition, pancreatic tumours are highly resistant to conventional types of therapy. Of the numerous anti-cancer agents investigated during the last decade, only erlotinib, an EGFR tyrosine kinase inhibitor has been added to the weaponry against pancreatic cancer. However, the therapeutic benefit of this approach is limited since the majority of patients simply do not respond or eventually acquire resistance to this type of therapy. The aim of this study was to investigate the anti-tumour effect of chemotherapeutic agents such as gemcitabine and several HER and IGF-IR inhibitors, including the pan-HER inhibitor afatinib, when used alone or in combination or 'in vitro'. The expression levels of all HER family members and IGF-IR in a panel of human pancreatic cancer and their association with response to treatment with HER and IGF-IR inhibitors was also investigated. Furthermore, the expression levels of these receptors as well as several ABC transporters and putative pancreatic cancer cell variants developed by chronic treatment with escalating doses of targeted agents or chemotherapeutic agent gemcitabine, of their parental counterparts. All pancreatic cancer cell lines investigated were found to be positive for EGFR, HER-2 and IGF-IR, however, over-expression of these receptors was found to be uncommon. In addition, no correlation was found between the expression levels of HER family members or IGF-IR and sensitivity to their respective inhibitors. Growth inhibition studies revealed that the pan-HER inhibitor afatinib had greater anti-tumour efficacy compared to first generation TKIs erlotinib and gefinitib, supporting its utilization in the treatment of this malignancy. Most importantly, co-targeting of HER family members and IGF-IR was found to be superior to single treatment, providing a rationale for investigating the therapeutic potential of this combination 'in vivo' (animal models and in a clinical setting).