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The anthraquinone di-N-oxide AQ4N is a prodrug designed to be excluded from cell nuclei until metabolised in hypoxic tumour regions to AQ4, a DNA binder and potent inhibitor of topoisomerase II. The antitumour effects of AQ4N in rodent neoplasms are well characterised but the identity of enzymes responsible for the metabolism are unknown. The aims of the present work were to identify Cytochrome P450 (CYP) enzymes responsible for AQ4N metabolism in rat and human tissue and to conduct a preliminary investigation into the in vivo metabolism of AQ4N in tumour bearing rodents. AQ4N was found to undergo a two electron reduction to the mono-Noxide AQM followed by a subsequent two electron reduction to cytotoxic AQ4. The process occurred in the microsomes of rat and human liver, was cofactor dependent and was inhibited by air. In rats, CYPs 2B and 2E were found to anaerobically metabolise both AQ4N and AQM. Kinetically, AQ4N metabolism conformed to a Michaelis-Menten model whereas the metabolism of AQM was better described by a sigmoidal relationship. In addition, both semi purified Cytochrome P450 reductase (CPR) and purified Nitric oxide synthase (NOS) were both able to anaerobically metabolise AQ4N. Both enzymes required NADPH and CPR mediated metabolism was dependent on the presence of exogenous haem. In humans, the anaerobic metabolism of both AQ4N and AQM correlated with CYP 3A activity and not with the activities of CYP 1 AI 2C and 2D. AQM metabolism correlated also with the activity of CYP 2A. The involvement of CYP 3A was confirmed by the use of CYP specific inhibitors and by the use of cDNA transfected cell microsomes. Human kidney and colonic tumours were found to anaerobically metabolise AQ4N and tumour metabolism was inhibited by the CYP inhibitor carbon monoxide (CO). Finally, the in vivo metabolism of AQ4N was studied in C3H tumour bearing mice. Metabolites of AQ4N were found in all tissues studied but the AQ4! AQ4N ratio was highest in the tumours. Collectively, these findings have identified the enzymes responsible for the metabolism of AQ4N and its mono-N-oxide. Differences exist between the CYP isoforms responsible for metabolism in rodents and in man, in humans, CYP 3A enzymes predominantly metabolise AQ4N and this subfamily of CYP are known to be well expressed in a broad spectrum of human cancers. With this in mind, AQ4N based therapy should be considered as a rational treatment regime for patients bearing solid tumour burdens.

The possibility of using elevated temperatures (hyperthermia) induced by ultrasound in cancer therapy has been investigated. Computer models were used to find the optimum frequency for the focussed ultrasonic transducer and lenses. It was found to be around 1 MHz when deep-lying tumours are to be treated. The effect of blood flow, thermal conduction and ultrasonic absorption on the temperature distributions during hyperthermia treatment was simulated with a 3-dimensional computer model. The decreased blood perfusion in some malignant tissues appeared to produce higher temperatures in the tumour than in the surrounding tissue despite equal energy input. The temperature distributions produced by focussed or overlapping fields were measured both in phantoms and in pigs' muscle in vivo. The thermal conduction smoothed the temperature distributions in both experiments. However, it was possible to produce the maximum temperature at the depth of 50 mm in muscle in vivo. By using 7 unfocussed overlapping fields the temperature of a large tissue volume could be increased. The biological effects of ultrasound hyperthermia were studied in transplanted rat's Yoshida sarcoma. The elevated temperature 49 °C was induced by a therapy transducer with the operating frequency of 0.75 MHz. The cell killing effect appeared to be similar to that reported by other research groups using different methods for induction of hyperthermia. Thus the most significant reason for the tissue damage in this experimental situation appears to be the thermal effect of ultrasound.

Malignant melanoma is resistant to almost all conventional forms of chemotherapy. Recent evidence suggests that anti-apoptotic proteins of the Bcl-2 family are overexpressed in melanoma and may contribute to melanomas striking resistance to apoptosis. ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-xl and Bcl-w, has demonstrated efficacy in several forms of cancer. However, overexpression of Mcl-1, a frequent observance in melanoma, is known to confer ABT-737 resistance. My results demonstrate that the combination of ABT-737 and RNA silencing of Mcl-1 induces significant cell death in six different melanoma cell lines, representing a potential new therapeutic strategy. I show that the apoptotic response to the combination treatment involves both the intrinsic pathway and a death receptor-independent role for extrinsic pathway proteins. The combination treatment also induces a number of gene expression changes as assessed by cDNA microarray and follow-up analyses. Based on the results of the array, I investigated the effects of inhibition of MAPK proteins combined with ABT-737 and/or Mcl-1 knockdown. I found that the combination of a p38 MAPK inhibitor and ABT-737 strongly and synergistically induces apoptosis in melanoma cell lines, thus suggesting a second novel treatment combination with potential for melanoma therapy. Finally, I provide novel evidence that Bcl-2 family member PUMA is cleaved in a caspase-dependent fashion during apoptosis and may play a role in treatment response. Currently, there are no effective treatments for metastatic melanoma. My findings describe two potential combination therapies for melanoma as well as provide novel evidence as to the mechanisms involved in treatment response.