No description available.

Despite intensive research, cancer remains one of the major causes of worldwide morbidity. It is widely believed, however, that if currently available anti-cancer drugs could be delivered specifically to tumours then the disease would have been mastered. The delivery of prodrug converting enzymes by clostridial spores specifically to the anoxic centres of tumours is one potential delivery mechanism. This is due to the extreme selectivity of spores to germinate solely in the hypoxic regions of tumours. Once germinated, the expression of a prodrug converting enzyme converts a systemical1y administered prodrug to a highly toxic drug only in the tumour. Previous studies using Clostridium acetobutylicum and Clostridium beijerinckii as the delivery vehicle highlighted that prodrug converting enzyme expression is only found in tumours. However, no significant anti-tumour affect was observed. Two possible reasons were evolved. Firstly, expression of the prodrug converting enzyme may be low, and/or, secondly, the tumours may not be colonised sufficiently to promote an antitumour effect. Preliminary studies identified that Clostridium sporogenes NCIMB 10696 may represent a more suitable host. Higher spore titres could be prepared and, once administered, higher cell counts are found in the colonised tumours. Prodrug converting enzymes with improved kinetics over pre-existing enzymes have also been identified. Once effective gene transfer systems and expression systems had been developed, suitably high levels of several different prodrug converting enzymes, in particular nitroreductases, were obtained. Initial in vivo studies on one of the early recombinant strains identified a definite anti-tumour effect. Since those initial studies, further improvements to expression have been made. It is hoped that a more significant anti-tumour affect would result from using these improved strains. It is the ultimate aim of CDEPT to have the prodrug converting enzymes integrated into the host genome so as to negate the use of antibiotics. Towards this, studies on the use of both classical and novel integrative technologies have been investigated.

No description available.

The diterpenoid paclitaxel (Taxol™) is one of the most effective anticancer drugs, used against a wide range of cancers. It is produced as a secondary metabolite in the vascular cambial region of the bark of Taxus brevifolia from which it was first extracted in 1971. Taxol also accumulates in low concentrations in several other Taxus species. As the demands for Taxol greatly exceeded its supply, alternative routes for producing the drug and its related taxanes were developed. Taxol is presently manufactured by semisynthesis from its precursors baccatin III and 10-deacetylbaccatin III found in Taxus needles. The biosynthesis of Taxol mostly occurs via the 2-C-methyl D-erythritol 4- phosphate pathway and requires at least 19 enzymatic steps from the precursor geranylgeranyl diphosphate. This study set out to heterologously express the early genes of the Taxol biosynthetic pathway in Nicotiana tabacum for the subsequent redirection of this precursor for the synthesis of novel taxanes. The first five genes of the Taxol biosynthetic pathway, namely taxadien-5-α-hydroxylase, taxadien-5α-acetyltransferase taxoids 10β, 13α- and 7β-hydroxylase, were isolated from Taxus baccata mRNA. Individual transgenic tobacco lines were generated expressing each of the first three enzymes of the biosynthetic pathway. These lines were crossed with each other in order to obtain all three transgenes expressed together in individual transgenic lines. Progenies from the crosses, expressing the first three transgenes were analysed, however, GS-MS analysis failed to detect the compound taxadiene-5α-ol and its acetylated compound taxadiene-5α-yl acetate. The expression of the Taxol biosynthetic genes in transgenic tobacco plants were accompanied by phenotypic effects, including dwarfism and low fertility of the transgenic plants. To circumvent these sterility issues which made crossing of the plants difficult, a construct was prepared carrying the first two genes of the Taxol biosynthetic pathway, to be transformed in yellow flesh tomato mutant at a later stage. The localisation of taxadiene synthase, 5α-hydroxylase and taxadien-5α acetyltransferase was investigated by making translational fusions to fluorescent protein tags. Confocal microscopy was used to detect the fluorescent proteins GFP, YFP and CFP in Arabiodopsis thaliana roots and tobacco leaf and root cells. Taxadiene synthase was found to be localised to the plastids, taxadien-5-α-hydroxylase spatially positioned on the plastid envelope and the endoplasmic reticulum membrane and taxadien-5α acetyltransferase was localised to the endoplasmic reticulum.

The study of the development of anti-cancer drugs and preclinical efficacy tests has until today encountered a major problem identified as lack of reliable in vitro tumour models which are able to reflect in vivo tumour conditions. These models provide a clear basis for understanding tumour development processes, assisting in the selection of agents from various chemicals and testing the efficacy of drugs. There are two important characteristics for an in vitro tumour model, i.e. tumour-like structure of cell aggregates, and the in vivo-like culture microenvironment. To meet these two requirements, an in vitro perfusion based three-dimensional tumour model was developed for the three dimensional culture of cancer cells and related anti-cancer drugs tests. In order to assess this model, DLD1 and NCI/ADR cells were cultured in four different models and compared their proliferation rate, cell viability, micro tumour formation and drug responses. In addition, the comparison of static and perfusion culture were done on monolayer and in 3D also. The cells in perfusion culture showed higher proliferation rates and significantly, higher cell viabilities after a 6-day culture compared to statically cultured cells, especially for the cells in the 3D culture. Microtumours (MTs) were formed from this model, which showed significant tumour-like morphological characteristics, a denser and highly stable structure, a higher cell viability, and varied drug responses compared with spheroids. The inhibition effect of paclitaxel and cisplatin, two common type anti-cancer drugs, were tested and a comparative study was carried out using conventional two-dimensional (2D) static culture, spheroids, and the developed 3D MTs model, as well as real human tumour tissues. The results showed that the cells in 2D culture were most greatly inhibited while human tumours showed the lowest drug responses. The efficacy of anti-cancer drugs, tested in conventional 2D static culture, was greatly amplified. Besides, the response of MTs to agents was much closer to that of human tumours, when the values of spheroids are relatively closer to the cells in 2D culture. It is further supported that MTs have more tumour-like characteristics than spheroids. When compared, the inhibition to proliferation of cells in static and perfusion culture showed significantly different drug responses except for the cells on the monolayer. The shown difference between static and perfusion culture can be due to the different culture environment, and further related to the different action mechanisms of anti-cancer agents. The perfusion culture provides a more homogenous and more physiological microenvironment for the in vitro tumour growth, and in vitro perfused 3D cancer model, developed in this thesis, proved valuable for the study of in vitro cancer and related anti-cancer drug tests.

Bibliography: leaves 144-163 / xi, 163 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (M.D.)--University of Adelaide, 1985

Cancer cells depend heavily on sustained expression of anti-apoptotic proteins. Targeting transcription to suppress these anti-apoptotic proteins seems a promising strategy for anti-cancer therapy. Cyclin-dependant kinase 9 (CDK9) regulates transcription elongation by phosphorylating Ser2 on the C-terminal domain of RNA polymerase II, while CDK7 phosphorylates Ser5 during transcription initiation. A screening cascade comprised of an MTT assay, a caspase-3 activation assay, a p53 stabilization assay and a mitotic index assay was developed to classify compounds and identify lead transcriptional CDK inhibitors from a novel class of 2,4,5-trisubstituted pyrimidines. Compounds S3-41 and CDKI-71 are the most potent CDK9 inhibitors identified by the screening cascade. They showed potent anti-proliferative activity in the MTT assay and induce both caspase-3 activity and p53 protein level at the GI50 concentration. In addition, no significant effect on mitotic index was observed. The detailed mechanism of action of CDKI-71 was further investigated and compared with the clinical compound, flavopiridol. Like flavopiridol, CDKI-71 displayed potent cytotoxicity and caspase-dependent apoptosis that were closely associated with the inhibition of RNAPII phosphorylation at Ser2. This indicated effective targeting of cyclinT-CDK9 and the downstream inhibition of anti-apoptotic proteins such as Mcl-l in cells. Similar to flavopiridol, CDKI-71 down-regulated a large range of genes, including Mcl-l and Bcl-2. No correlation between apoptosis and inhibition of cell cycle CDKs 1 and 2 was observed. Non-transformed lung fibroblast cell lines showed resistance to CDKI-71 treatment. In contrast, flavopiridol showed little selectivity between cancer and normal cells. Flavopiridol also induced genotoxic stress through the induction of DNA double-strand breakage. These results suggest that CDKI-71 has a great potential to be developed as an anti-cancer agent. Another study focused on in vitro anti-tumour mechanism of CDKI-83, a dual inhibitor of CDK9 and CDKI, was performed in A2780 ovarian cancer cells. CDKI-71 presented potent anti-proliferation activity and induced apoptosis in A2780 cells. By inhibiting cellular CDK1 and CDK9 activities, CDKI-83 arrested cells in G2 phase and reduced anti-apoptotic proteins at both mRNA and protein levels, respectively. This study suggests that the combination of CDK9 and CDK1 inhibition results in effective induction of apoptosis in cancer cells.

Bibliography: leaves 144-163 / xi, 163 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (M.D.)--University of Adelaide, 1985

Lactate Dehydrogenase-A (LDH-A) is up-regulated in a broad array of cancers and is associated with poor prognosis. Involved in the hypoxic response, LDH-A is a HIF-1 target and is responsible for the enzymatic reduction of pyruvate to lactate. This is important for several reasons, chiefly (1) the regeneration of NAD+ which feeds back into earlier glycolytic stages and (2) the depletion of intracellular pyruvate concentrations. High intracellular pyruvate is known to inhibit HDACs and is associated with increased apoptosis. LDH-A is also known to be controlled by oncogenes such as c-Myc suggesting an oncogenic role. Studies have shown that the knock-out of LDH-A reduces proliferation and tumourgenicity, and stimulates the mitochondria. This thesis therefore had three aims: firstly, to validate LDH-A inhibition and elucidate its full nature in terms of the implications for tumour survival; secondly, to ascertain the role of LDH-B in order to determine whether selectivity towards LDH-A would be a necessary feature of any small molecule; lastly, to recapitulate siRNA mediated LDH-A inhibition with small molecule inhibitors that had the potential for clinical application. The thesis examined both clinical data and a broad panel of cultured cancer cell types in order to select appropriate model in which to validate siRNA mediated inhibition of LDH-A and LDH-B. After it was demonstrated that LDH-A inhibition reduced the growth of cultured cells, a range of techniques were used to quantify this reduced growth in terms of cell death and changes in metabolism. Further to this, literature studies had proposed a role for LDH-B in maintaining lactate fuelled tumour growth; however, this thesis shows that in the cell lines studied, lactate-fuelled tumour growth was an LDH-A dependent phenomenon. Finally, a high throughput assay system was designed and validated and a library of small molecules was selected, synthesized, and screened in order to identify selective inhibitors of LDH-A.

The aim of the work described in this thesis was to explore the synthesis of glycosidase inhibitors and to evaluate their potential as anti-cancer agents. Glycosidases catalyze the fission of glycosidic bonds and are involved in vital biological functions. With regard to their potential for anti-cancer therapy, two glycosidases were identified: α-N-acetyl-galactosaminidase and β-N-acetyl-hexosaminidase. The former has been implicated in causing immunosuppression in advanced cancer patients by negating the effect of the macrophage activating factor (MAF), while the latter is secreted by invading cancer cells and hence associated with metastasis formation. The synthetic focus was on generating piperidine and azetidine iminosugars, carbohydrate mimetics with their endocylic oxygen replaced by nitrogen. Their structural similarity to carbohydrates make iminosugars excellent inhibitors of glycosidases. Following synthesis of a pipecolic amide, its previously reported potent β-N-acetyl-hexosaminidase inhibition was confirmed. This data, along with inhibition profiles of several pyrrolidines, allowed the generation of a molecular model for predicting activity of β-N-acetyl-hexosaminidase inhibitors. The model was used to select azetidines in the D/L-ribo and D-lyxo configuration as suitable candidates to be explored in novel chemical space, leading to the first synthesis of a fully unprotected 3-hydroxy-2-carboxy-azetidine. The potent α-N-acetyl-galactosamindase inhibitor 2-acetamido-1,2-dideoxy-D-galacto-nojirimycin (DGJNAc) was successfully derivatised via N-alkylation. Important structural discoveries with regard to glycosylation of vitamin D₃-binding protein, the precursor of MAF, were made using MALDI mass-spectrometry. By comparing the enzymatic and cellular inhibition of N-alkylated derivatives of DGJNAc and a pyrrolidine the following generalization on iminosugar biodistribution was found: N-butylation promotes uptake into the cell/organelles, while hydrophilic side-chains restrict cellular access. An in vitro assay evaluating cancer cell invasion was devised and β-N-acetyl-hexoamindase inhibitors were shown to retard cell migration, including with the highly metastatic breast cancer cell line MDA-MB-231. Additive effects where found when the iminosugar was combined with a protease inhibitor, suggesting potential for future combination therapy.