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In comparison to other cancers, advanced prostate cancer is resistant to chemotherapy. There is a need to understand the mechanisms which are responsible for this resistance and find better treatments for this disease or methods to increase the efficacy of current treatments. Cancer cells often evade apoptosis. Cyclooxygenase-2 (COX-2) is an enzyme reported to be elevated in prostate cancer, and has oncogenic properties, including apoptosis attenuation. Because of this, COX-2 inhibition could be beneficial for both prevention and treatment of cancer. This study showed that COX-2 protein was not detected in LNCaP, PC-3 or DU145 cells. To assess the effect COX-2 has on the apoptotic sensitivity of prostate cancer cells, we transfected two cell lines (stable transfection in LNCaP, transient in PC-3) with the human COX-2 gene or empty control vector. We measured the effect on cell viability of COX-2 after treatment with a diverse set of agents e.g. etoposide, carboplatin, Fas, TRAIL, celecoxib and sulindac, using the MTT assay. We observed COX-2 dependent resistance to carboplatin, etoposide and celecoxib in LNCaP but not PC-3. Carboplatin mediated reduction in cell viability was due to an S phase block and induction of apoptosis. COX-2 transfection in LNCaP cells attenuated both the cell cycle block and apoptosis. There was reduced p53 and p27KIP1 induction following carboplatin treatment in LNCaP-COX-2, compared to LNCaP-Neo. COX-2 transfection caused elevated cellular- levels of anti-apoptotic proteins Bcl-2, BC1-XL and survivin. Summary Celecoxib could not reverse the resistance seen in LNCaP-COX-2 to carboplatin, and PGE2 could not increase the resistance in LNCaP-Neo cells, suggesting that COX-2 mediates an apoptotic resistance which is COX-2 enzymatic activity independent. Other mechanisms were sought to reverse the carboplatin resistance. PI3K inhibitors wortmannin and LY294002 partially reversed the LNCaP-COX-2 resistance. These data suggests that COX-2 acts on the PI3K signalling pathway to mediate resistance in LNCaP. This was confirmed by Western blot findings of elevated levels of in P-Aktser473 LNCaP-COX-2 compared with LNCaP-Neo. Celecoxib decreased levels of P-Aktser473 inn a manner similai- to PI3K inhibitors, indicating that the PI3K inhibitors and celecoxib act in different ways on PI3K signalling. Because complete reversal of resistance does not occur with PI3K inhibition, COX-2 must be acting on other pathways also. However, unlike the PI3K inhibitors, celecoxib could not reverse resistance to carboplatin. Wortmannin and LY294002 decreased levels of Bcl-2, BC1-XL, survivin and, surprisingly, COX-2 protein. In contrast, celecoxib had little effect on Bcl-2 and increased COX-2 levels. Cyclin B1 levels were higher in LNCaP-COX-2 than LNCaP-Neo. Gene microarray analysis confirmed the up-regulation of survivin in LNCaP-COX-2 and also showed elevation of cyclin I and fatty acid synthase and down-regulation of glutathione-S-transferase in LNCaP-COX-2. In conclusion, stable COX-2 transfection causes a selective resistance to cytotoxic agents which is mediated via activation of the PI3K pathway and attenuation of p53 induction in LNCaP. COX-2 also causes the up-regulation of a number of anti- apoptotic factors and PI3K inhibition results in down-regulation of these proteins.

Sphingosine kinase 1 (SPHK1) is an oncogenic enzyme that is upregulated in a wide range of human tumours and is associated with cancer progression. The product of SPHK1's activity, sphingosine-1-phosphate (S1P) enhances metastatic potential by promoting cancer cell migration and invasion. Our group has previously reported that in vitro SPHK1 inhibition potentiates the effects of docetaxel, a key treatment of prostate cancer (PCa) , implicating a potential therapeutic role of SPHK1. The work presented in this thesis investigated the mechanism for docetaxel-induced apoptosis in PC-3 cells and demonstrated the involvement of a SPHK1-dependent and -independent pathway. The dose-dependent inhibition of SPHK1 by docetaxel led to an initial loss of enzyme activity followed by a decrease in SPHK1 expression. Further, in prostate cancer cell models, SPHK1 inhibition had a significant chemosensitising potential leading to a 4-fold reduction in the effective dose of docetaxel. To link SPHK1 upregulation in prostate cancer cells to potential targets, this thesis investigated the nature of the interaction between SPHK1 and prosaposin signalling pathways. Prosaposin is a neurotrophic secreted protein involved in cancer progression and chemoresistance. Recent reports suggest that prosaposin activates SPHK1 in cancer cells. The work presented in this thesis demonstrates that prosaposin knockout mice exhibited a significant reduction in SPHK1 activity in the prostate and seminal vesicles. In prostate cancer cell lines, SPHK1 activity correlated with the amount of secreted, but not intracellular prosaposin. This suggests a role of the prosaposin/G-protein coupled receptor (GPCR) signalling. Blocking prosaposin in prostate cancer cells induced a signi cant decrease in SPHK1 activity and expression. Conversely, exogenous prosaptide TX14A, derived from the trophic sequence of saposin C, enhanced SPHK1 activity and expression. In turn, SPHK1 activation is essential for prosaposin secretion, but not for its expression. Increased SPHK1 expression or exogenous S1P triggered prosaposin secretion, while inhibition of SPHK1 using speci c siRNA or pharmacological inhibitor drastically decreased prosaposin secretion. Both prosaposin and SPHK1 signalling pathways were shown to be involved in the production of cytokines and angiogenic factors such as plasminogen activator inhibitor-1 (PAI-1), macrophage migration inhibitory factor (MIF) and pentraxin-3 (PTX3). These findings were extended to the clinical situation, and circulating prosaposin levels were determined in prostate cancer patients and compared with healthy controls. Increased prosaposin serum levels correlated with tumour stage. The work in this thesis has shown for the rst time the cross-regulation of prosaposin and SPHK1 in prostate cancer. The mechanism and physiological relevance of this cross-regulation in prostate cancer cells has been investigated and found to have significant therapeutic and biomarker potential in advanced prostate cancer.

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Conventional treatments for prostate cancer have significant limitations making it difficult to control the disease. Cyclopropabenzindoles (CBI) are more biologically potent, stable and synthetically accessible analogues of cyclopropapyrroloindole (CPI) anti-tumour antibiotics, such as duocarmycin-SA and CC1065. A polymeric prodrug carrying a CBI drug attached to the polymeric backbone through a PSA cleavable linker peptide has two modes of selectivity: activation by PSA and the EPR effect. To synthesise a 5-amino-seco-CBI analogue, 2,4-dinitronaphthalen- 1-ol gave di-Boc-1-iodonaphthalene-2,4-diamine in five steps (triflation, SNAr displacement with iodide, reduction (loss of iodine), protection and restoration of the iodine. For the amino-seco-CBI, it was important to discriminate between N2 and N4. Acidic removal of the Boc-group(s) resulted in deiodination. NMR investigations showed an unexpected Wheland-like cationic intermediate. N3 of naphthalene-1,3-diamine was selectively trifluoroacetylated and N1 was masked with Boc. Electrophilic iodination gave an orthogonally protected 1-iodonaphthalene-2,4-diamine. Allylation at the trifluoroacetamide was followed by free radical cyclisation with TEMPO trap. Removal of the trifluoroacetyl group allowed coupling to 5-(2-(dimethylamino)ethoxy)-1H-indole-2-carboxylic acid. Reductive removal of 2,2,6,6-tetramethylpiperidine, substitution of the exposed hydroxy group with chloride and removal of the Boc-group gave the amino-seco-CBI drug, 5-amino-1-chloromethyl-3-(5-(2-dimethylaminoethoxy)indole-2-carbonyl)-2,3-dihydro-1H-benz[e]indole. A DNA-melting assay confirmed that it binds very strongly to dsDNA causing a 13 deg. C increase in melting temperature. The drug was a highly potent cytotoxin in vitro, with IC50 = 18 nM against LNCaP prostate cancer cells. The polymeric prodrug system involved the synthesis of the pentapeptide SSKLQ. The amide side chain of glutamine can be masked as the nitrile and this can be quantitatively hydrated to the γ-carboxamide of L-Gln with hydroperoxide. The pentapeptide was coupled to 4-methoxynaphthalen-1-amine and to poly(ethylene glycol) as a model polymeric prodrug system. Efficient release of the model drug from the polymeric prodrug by PSA will allow this polymeric prodrug system to be adopted for the synthesised amino-seco-CBI drug.