published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Platinum-based anticancer drugs such as cisplatin, carboplatin and nedaplatin have been widely used in the chemotherapy of a variety of solid tumours for several decades. However, the development of both inherent and acquired resistance has greatly limited the efficacy of all of these drugs. Several mechanisms were proposed to explain the cellular resistance to these platinum drugs, including decreased drug accumulation. Previously, it was suggested that cisplatin enters cells via passive diffusion, followed by intracellular hydrolysis and activation prior to targeting DNA. However, recent in vivo and in vitro studies confirmed that transporters and carriers involved in copper homeostasis play important roles on the transport as well as cellular resistance to the platinum drugs. CTR1, a major plasma-membrane transporter involved in intracellular copper(I) homeostasis, was found to facilitate the uptake of several platinum drugs although the molecular mechanism remains unclear. The extracellular N-terminal domain of human CTR1 (hCTR1) with two methionine(Met)-rich and two histidine(His)-rich motifs has been proved to be essential for the uptake of both copper and platinum drugs by the transporter. In this thesis, the extracellular domain of hCTR1 (hCTR1_N, residues 1-55) was overexpressed and the role of the Met- and His-rich motifs on cisplatin binding was examined by either mutagenesis or chemical modification. Cisplatin was found to directly and rapidly bind to the Met residues of hCTR1_N by the formation of monofunctional cisplatin-thioether adducts. The kinetics of the binding process was found to correlate with the number of Met residues, indicating that all Met residues are exposed to solvents and capable for cisplatin binding. Such a non-sequence-specific binding may increase the likelihood of capturing the anticancer drug in extracellular fluid by the N-terminus of hCTR1. The effect of hCTR_N on the binding and activation of second-generation platinum anticancer drugs, e.g. carboplatin and nedaplatin, were subsequently investigated. hCTR1_N was found to significantly facilitate the activation of these platinum drugs by the formation of ring-opened monofunctional Pt-thioether species through Met residues. Although the activities of platinum drugs against hCTR1_N are significantly different, their monofunctional protein-bound species demonstrated great similarity in both structure and kinetic aspects, suggesting the uptake of these platinum drugs by hCTR1 might follow the same mechanism. The formation of active ring-opened species of carboplatin and nedaplatin by chloride/bicarbonate was observed, indicating these nucleophiles may play a critical role in the pre-activation of the drugs prior to their reaching cellular targets. Pt-thioether species were proposed as intermediates for the platination of other biomolecules. The monofunctional cisplatin adduct of hCTR1_N was proved to further transfer its active platinum species to either cysteine- or guaninecontaining biomolecules which mimic the C-ternimus of hCTR1 and DNA. Methionine residues of hCTR1 may therefore serve as key residues for the activation and transport of platinum anticancer drugs in the form of monofunctional Pt-thioether species through the pole of trimeric hCTR1 and eventually to their final target – DNA. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

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Experiments were carried out to examine the value of CCS (copper chaperone for CuZn superoxide dismutase) as a novel biomarker of zinc-induced mild copper deficiency and to evaluate the changes in expression of zinc transporters in response to graded levels of moderately high dietary zinc. Weanling male Wistar rats were fed graded levels of zinc (30, 60, 120 and 240 mg zinc/kg diet) for 5 weeks. Results showed a dose-dependent decrease in copper content and an increase in CCS expression in tissues of rats fed the Zn-60 and Zn-120 diets. Surprisingly, rats fed the Zn-240 diet showed better copper status than rats fed the Zn-120 diet. Expression of zinc transporters was significantly upregulated in the small intestine of Zn-240 rats. Collectively, these data show that CCS is responsive to zinc-induced mild copper deficiency, and can serve as a sensitive biomarker of mild copper deficiency. The increased expression of intestinal zinc transporters expression may account for the better copper status of Zn-240 rats.