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.