It has been shown that the majority of benign prostatic hyperplasia (BPH) and prostate cancers are dependent on androgen production within the body. The biosynthesis of androgens is catalysed by different enzymes however one of the enzymes, 17 beta-hydroxysteroid dehydrogenase type 3 (17 beta-HSD3), converts the C(17)=O carbonyl moiety of androstenedione (1l4-dione) to the corresponding C(17)-OH hydroxyl group of testosterone (T). It has been hypothesised that inhibition of 17 beta-HSD3 may cause a decrease in the level of androgens which in turn leads to a reduction in the genesis of androgen-dependent prostatic diseases. The utilisation of enzyme inhibition as a therapeutic agent, in the treatment of breast cancer, has been tested on postmenopausal women by using aromatase inhibitors (e. g; exemestane, anastrazole and latrazole). This approach has proved to be successful and the impact of enzyme inhibition was led to a reduction in cancer growth. This process has now found a clinical application. From molecular modelling studies it was postulated that any potential inhibitor of 17 beta-HSD3 should contain a carbonyl moiety, mimicking the C(17)=O of the natural substrate, as well as an aromatic ring adjacent to the carbonyl group. With these criteria in mind results from our laboratories showed that from a library of candidates those based upon 4-hydroxyphenyl ketones showed some potential. The main focus of this prestn study was to fine tube the enzyme inhibitor analogues and hence optimise inhibitory activity of 4-hydroxyphenyl ketones. We have successfully synthesised a range of novel derivatives of 4-hydroxyphenyl ketones such as the 4-methanesulfonate and 4-acetate ester derivatives. In general, the reactions have proceeded very well with the yields ranging from 65% to 88% and 91% to 97% respectively. The results of biochemical evaluation studies suggested that the acetate ester derivatives, in particular compounds (149) and (150) exhibited good inhibitory activity against 17 beta-HSD type 3 of about 40% compared to standard inhibitors such as 7-hydroxyflavone and baicalein which resulted in about 13% and 14% inhibitory activity respectively. In addition a range of non-steroidal B, C, D ring mimics of the natural substrate of 17 beta-HSD type 3 were synthesised in good yields (65% to 85%). The biochemical evaluation of these compounds also showed good inhibitory activity; in fact compound (107) exhibited about 43% inhibition in comparison to the above standards which had inhibition of about 25% and 31 % respectively. In conclusion we have successfully synthesised and biochemically evaluated a number of enzyme inhibitors for the enzyme 17 beta-HSD type 3. The two types of active inhibitors were structurally dissimilar suggesting that they may have different modes of binding. This outcome requires further investigation in order to establish and identify how this inhibition is taking place.

Androgens play an important part in the initiation and progression of hormone-dependent prostate and breast cancer. These types of cancers can be treated by androgen ablation therapy. However, androgen ablation is associated with short (2-3 years) remission of the disease. Therefore therapies that inhibit the systemic biosynthesis of androgens, by targeting the P450 enzymes (CYP17 and 17-[beta]HSD type 3) which catalyse androgen biosynthesis, may represent a rational approach in the treatment of androgen-dependent cancer. Inhibitors of the enzyme CYP17: ketoconazole and liarozole, have been shown to decrease tumour cell adhesion to the endothelium and expression of adhesion molecules. The adhesion of cancer cells to the endothelium is an important preliminary event that underlies cancer matastasis. Within the this study, the development of assays for the enzymes; CYP17 and 17[beta]-HSD3 and the evaluation of a series of compounds which were designed to inhibit these enzymes have been considered. The preliminary screening of the compounds showed good inhibition of 17[alpha]=OHase and 17, 20 lyase components of the CYP17 enzyme in comparison to the reference drug, ketoconazole (KTZ). The IC[sub]50 of compounds 31, 34, 38, 41, 48 and 51 and KTZ was calculated as 14.40 [Mu]M, 5.82 [Mu]M, 0.18 [Mu]M, 1.35[Mu]M, 1.21[Mu]M, 0.50[Mu]M and 5.65[Mu]M respectively. However, only a few of the compounds designed to inhibit 17[beta]-HSD3 showed ap potent inhibitory activity. Compound 132 showed the highest percentage inhibition (40.51 [plus or minus] 0.14%) of 17[beta]- HSD3 activity when compared to the reference drugs, 7-hydroxy flavone (12.90 [plus or minus] 0.31%) and biacalein (13.66 [plus or minus] 0.31%). CYP17 inhibitors did not have any cytoxic effect on human cancerous and non-cancerous cell lines. The adhesion of DU145, PC3 and MCF7 to a non-stimulated HUVEC monolayers was decreased from 100 [plus or minus] 0.01% cell adhesion to 60.93 [plus or minus] 3.95%, 65.79 [plus or minus] 9.39% and 65.12 [plus or minus] 4.04% by compounds 38. 48 and 51 respectively in the absence of tumour necrosis factor alpha (THF-[alpha]). Similarly, compounds 38, 48 and 51 showed the highest anti-adhesion effect of DU145 on stimulated HUVEC monolayers (69.85 [plus or minus] 2.51%) cells respectively. Flow cytometry and immunostaining of intracellular adhesion molecules showed that CYP17 inhibitors did not have any effect on the expression of ICAM-1. In conclusion, the synthesised compounds were found to be good indicators of the CYP17 enzyme with no cytoxic and better anti-adhesion effects when compared to KTZ. Thus, these compounds can be further investigated as a therapeutic strategy against hormone-dependent prostate and breast cancer.

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.

Despite the significant improvement in CML treatment since the introduction of Glivec, resistance to synthetic Tyrosine Kinase Inhibitors is emerging as a major limitation. 50%-90% of patients acquire resistance through point mutations that might span the ABL1 kinase domain, while 10%-50% may resist treatment through other mechanisms. Qatar with its limited 1.6 million inhabitants and 15 CML patients diagnosed yearly has the highest rate of disease resistance to Glivec treatment. Through a prospective study, this project examined the rate of resistance to Glivec and the different mechanisms that could be responsible for this problem. Thus over a period of 5 years, 26 patients were treated with Glivec as a front line therapy and their response to treatment was monitored objectively according to the ELN 2006 response criteria. 12 of the 26 patients responded optimally to treatment, while 14 patients did not (9 failed the treatment and 5 responded sub-optimally), setting the resistance rate to about 54% which is the highest reported worldwide to date. None of our patients showed any of the reported mutations that are known to confer resistance to Glivec. However in one patient, direct sequencing showed a Single Nucleotide Polymorphism (SNP) that might function as a resistance inflecting mutation. In another patient who resisted treatment a transient insertion of three nucleotides (AAG) at position 1432 which adds an amino acid Lysine to position 356 of the catalytic domain of ABL1 was revealed. To our knowledge this transit insertion of nucleotides and amino acid addition was not reported before. 7 patients showed Additional Chromosomal Abnormalities (ACA) at time of resistance, while 2 patients were intolerant to treatment and 3 had no identifiable cause for their resistance. Patient compliance was ruled out as a cause of resistance in our patients, however the quality of service providing was audited and certain financial and clinical management issues were addressed during the course of this study.

Adoptive Cell Therapy of cancer using T cells is entering mainstream practice after years as a research method. Central to the efficacy of this “living therapy” is the function of the T cells transferred. T-cells, like other primary tissue cells, undergo differentiation and death. Clinical and preclinical data shows that lesser differentiated, less glycolytic, and more proliferative-capable cells used for the adoptive transfer yield superior tumor responses. This introductory section will describe discoveries which elucidate and control T-cell differentiation and function for the improvement of adoptive cell therapy. Namely by use of inhibition of the PI3k/AKT pathways, and through the discovery of a dual function of death and differentiation by the canonical death receptor Fas, which can be parsed apart with a mutation from valine to cysteine at the 194 position (C194V), differentiation can be withheld while leaving cell proliferation unhindered during T-cell stimulation and expansion. The data also reveals that the differentiation signal caused by extracellular Fas ligation passes through AKT, revealing both Fas and AKT as points of intervention for targeting differentiation along the same pathway. From further investigation, this introduction will describe the effect of AKT inhibition on T-cell differentiation on a transciptional and metabolic level. The data reveals AKT inhibition promoted FOXO1 intranuclear organization, which creates a more naive-like phenotype to the cells, and lower glycolytic status, another phenotype associated with persistent and long-lived cells. Furthermore, this control of AKT and Fas in T-cells yields benefits in several modalities of pre-clinical models of adoptive T-cell immunotherapy of cancer, in both use of a chimeric antigen receptor (CAR) and with use of Tumor Infiltrating Lymphocytes (TIL). Finally, the real-world applicability of the finding including the use of AKT inhibition in current approved Adoptive T-cell immunotherapies will be discussed.

In this dissertation, we are interested in studying processes which generate events repeatedly over the follow-up time of a given subject. Such processes are called recurrent event processes and the data they provide are referred to as recurrent event data. Examples include the cancer recurrences, recurrent infections or disease episodes, hospital readmissions, the filing of warranty claims, and insurance claims for policy holders. In particular, we focus on the multi-type recurrent event times which usually arise when two or more different kinds of events may occur repeatedly over a period of observation. Our main objectives are to describe features of each marginal process simultaneously and study the dependence among different types of events. We present applications to a real dataset collected from the Nutritional Prevention of Cancer Trial. The objective of the clinical trial was to evaluate the efficacy of Selenium in preventing the recurrence of several types of skin cancer among 1312 residents of the Eastern United States. Four chapters are involved in this dissertation. Chapter 1 introduces a brief background to the statistical techniques used to develop the proposed methodology. We cover some concepts and useful functions related to survival data analysis and present a short introduction to frailty distributions. The Monte Carlo expectation maximization (MCEM) algorithm and copula functions for the multivariate variables are also presented in this chapter. Chapter 2 develops a multi-type recurrent events model with multivariate Gaussian random effects (frailties) for the intensity functions. In this chapter, we present nonparametric baseline intensity functions and a multivariate Gaussian distribution for the multivariate correlated random effects. An MCEM algorithm with MCMC routines in the E-step is adopted for the partial likelihood to estimate model parameters. Equations for the variances of the estimates are derived and variances of estimates are computed by Louis' formula. Predictions of the individual random effects are obtained because in some applications the magnitude of the random effects is of interest for a better understanding and interpretation of the variability in the data. The performance of the proposed methodology is evaluated by simulation studies, and the developed model is applied to the skin cancer dataset. Chapter 3 presents copula-based semiparametric multivariate frailty models for multi-type recurrent event data with applications to the skin cancer data. In this chapter, we generalize the multivariate Gaussian assumption of the frailty terms and allow the frailty distributions to have more features than the symmetric, unimodal properties of the Gaussian density. More flexible approaches to modeling the correlated frailty, referred to as copula functions, are introduced. Copula functions provide tremendous flexibility especially in allowing taking the advantages of a variety of choices for the marginal distributions and correlation structures. Semiparametric intensity models for multi-type recurrent events based on a combination of the MCEM with MCMC sampling methods and copula functions are introduced. The combination of the MCEM approach and copula function is flexible and is a generally applicable approach for obtaining inferences of the unknown parameters for high dimension frailty models. Estimation procedures for fixed effects, nonparametric baseline intensity functions, copula parameters, and predictions for the subject-specific multivariate frailties and random effects are obtained. Louis' formula for variance estimates are derived and calculated. We investigate the impact of the specification of the frailty and random effect models on the inference of covariate effects, cumulative baseline intensity functions, prediction of random effects and frailties, and the estimation of the variance-covariance components. Performances of proposed models are evaluated by simulation studies. Applications are illustrated through the dataset collected from the clinical trial of patients with skin cancer. Conclusions and some remarks for future work are presented in Chapter 4. / Ph. D.