The emergence of multi-drug resistance (MDR) to antimalarial and anticancer drugs has stimulated a search for novel MDR inhibitors/reversers. Bisbenzylisoquinoline alkaloids (BBIQ) are potential agents for reversing MDR, especially when used as synergistic enhancers of anticancer and antimalarial drugs with improved therapeutic efficacy. Despite numerous useful biological activities reported for BBIQ’s, the various syntheses of individual members remained cumbersome and the overall yields are low. In addition, published methods are nonstereospecific and produced racemates. The aim of this project was to develop a synthetic pathway for the preparation of cycleanine, a natural BBIQ with a symmetrical structure. The protocols developed for the synthesis of cycleanine will serve as a template for the synthesis of other BBIQ’s with more complex structures. The only published total synthesis of cycleanine did not address regioselectivity and stereoselectivity, furthermore, key steps suffered from extremely low yields of the products. Our synthetic pathway is a chiral auxiliary-based asymmetric synthesis that generates enantioselectively a 1,2,3,4-tetrahydroisoquinolines (THIQ) monomers. Cheap, commerciallyavailable starting materials were used to prepare monomers in a regioselective as well as stereoselective manner in good yields. The key feature of this method entails coupling of a chiral β-phenethylamine and halophenylacetaldehyde using the Pictet-Spengler reaction. Due to the difficulties encountered during the course of the preparation of monomers, different methods were tried and formation of unanticipated products rationalised. Dimeric BBIQ’s are constituted of monomeric THIQ’s which are reported to have array of biological properties including MDR reversing activities, therefore, the total synthesis of cycleanine will serve two purposes. In this investigation, the THIQ monomers were synthesised by a pathway that avoid harsh reaction conditions. Major reactions employed include nucleophilic aromatic substitutiton, Wittig reaction, hydroboration and IBX oxidation. Some of the steps were attempted on model compounds to optimise the conditions prior to attempting the reaction on cycleanine precursors. Two major contributions toward the synthesis of BBIQ’s were made in this study. The reaction conditions to control the regioselectivity and enantioselectivity of the Pictet- Spengler reaction for the preparation of THIQ moiety were developed. A major drawback of the published syntheses of BBIQ’s is the harsh conditions and low yields associated with the Ullmann reaction, which is used in the formation of the diaryl ether bonds. We have shown that the microwave-assisted nucleophilic aromatic substitution of aryl fluorides provide a much superior method for the formation of the key diaryl ether bond. Although we failed to form the final diaryl ether bond, the pitfalls encountered in the synthetic pathway are discussed and potential solutions are presented. The developed synthetic pathways are of general applicability and therefore can also be employed in the synthesis of other macrocyclic natural products containing diaryl ethers. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Small cell lung cancer (SCLC) is an aggressive form of lung cancer that frequently develops multidrug resistance (MDR) during chemotherapy. Vault RNA1 (vRNA1), a non-structural component of the MDR-associated vault organelle, is believed to act as a microRNA (miRNA) and may contribute to MDR by regulating the expression of genes involved in apoptosis, inflammation, and/or drug metabolism. Since vaults function to aid cells in survival, we hypothesized that vRNA1 might be free in the cytoplasm and able to inhibit expression of pro-survival mRNAs when vaults are open in drug-sensitive GLC4/S cells but not in the MDR GLC4/ADR cells where vaults might be closed with the miRNA sequestered within. In order to establish the role of vRNA1 as a regulator of survival in SCLC cells, siRNA-mediated down-regulation of vRNA1 was employed in GLC4/S and GLC4/ADR SCLC cells. Fluorescence microscopy using a green fluorescent 3’ AlexaFluor-488 negative siRNA control was used to estimate transfection efficiency, yielding 56% for GLC4/S and 89% for GLC4/ADR. However, these values and the level of apoptosis before and after transfection, as judged by trypan blue hemacytometer cell counts, were not entirely reliable due to cell clumping. The latter counts indicated a 2-fold decrease in viability in GLC4/S cell following transfection but no decrease in GLC4/ADR cells (p< 0.05). RT-PCR revealed that transfection significantly (p<0.05) decreased vRNA1 expression in GLC4/S cells but not in GLC4/ADR cells, confirming our hypothesis concerning the availability of vRNA1 in the two cell types. Caspase activity measurements showed vRNA1 down-regulation in the GLC4/ADR cells significantly (p≤0.05) increased survival via a 6.1-fold reduction in caspase 3/7 activity, further supporting our hypothesis. However, GLC4/S cells showed a similar loss of apoptosis when transfected with either sivRNA1 or the negative control siRNA. vRNA1 down-regulation did not significantly (p≤0.05) affect the expression of major pro-survival (Bcl-2, Bcl-xL), pro-apoptotic (Bad), or pro-inflammatory (IL-6, NFĸB p65) factors in either GLC4/S or GLC4/ADR cells. However, the drug metabolism protein CYP3A (previously shown by Persson et al., 2009 to be regulated by vRNA1) was significantly (p≤0.05) lowered (~16%) following vRNA1 down-regulation in the GLC4/S cells. In conclusion, we were successful in down-regulating vRNA1 which enhanced cell survival as hypothesized, but we were not able to identify new proteins regulated by vRNA1. / Department of Biology

Only discovered about 20 years ago, the structure of the eukaryotic vault particle has been studied extensively, but the function has yet to be determined. Vault numbers are up regulated in many types of cancer cells that are treated with chemotherapy agents and it is thought that they may act to transport chemotherapy drugs out of such cells, leading to multi-drug resistance (MDR). To determine a possible role of the vault particle in MDR, the goal of this research was to examine one of the functional vault proteins, vault poly(ADP)ribose (VPARP) for interactions with other proteins. Two forms of small cell lung cancer cells were used; GLC4/S which do not exhibit MDR and the MDR cells GLC4/ADR, which are cultured with the chemotherapy drug doxorubicin. Both cell cultures were subjected to a subcellular fractionation followed by gentle immunoprecipitation with an antibody to VPARP. Immunoprecipitated proteins interacting with VPARP were only observed in GLC4/ADR cells, as seen on a PAGE gel. This sample was taken to Monarch Life Sciences and analyzed by mass spectrometry. One interacting protein was found to be NALP1 pyrin domain (PYD), a member of the death domain family of proteins which is involved in inflammation and apoptosis. The interaction of VPARP with NALP1, which only occurred in MDR cells, suggests an exciting, previously unreported possibility – that VPARP binding may inhibit NALP 1-stimulated apoptosis when MDR is occurring. Future studies are needed to examine if levels of NALP1 vary in GLC4 cells with and without treatment with doxorubicin and in normal lung cells. The cellular location (nucleus or cytoplasm) of the interactions should also be identified. Furthermore, immunoprecipitation of proteins interacting with NALP1 should include VPARP and perhaps identify other proteins interacting in the signaling pathways under MDR and normal culture conditions. This information may contribute insight into the function of VPARP and vaults within the cell. / Department of Biology

Multidrug resistance refers to the simultaneous resistance to structurally and mechanistically unrelated cytotoxic drugs. Chronic administration of cytotoxic drugs to patients with metastatic breast cancer results in the development of multidrug resistance, thus rendering chemotherapy unsuccessful. One mechanism by which multidrug resistance is conferred is the decreased intracellular drug accumulation due to the upregulation of the ATP-binding cassette (ABC) transporters. Jadomycins are polyketide-derived natural products produced by the soil actinomycetes Streptomyces venezuelae, ISP 5230. Jadomycins exhibit anticancer, antibacterial and antifungal activities. Pilot work in our laboratory demonstrated that jadomycin B exhibited similar cytotoxic effects in drug-sensitive and drug-resistant cancer cells. We hypothesize that jadomycins are poor substrates of ABCB1, ABCC1 and ABCG2 efflux transporters, and consequently will exhibit higher intracellular accumulation, which results in improved cytotoxic efficacy over existing chemotherapeutics that are rapidly effluxed by ABC transporters. Using methyltetrazolium (MTT) cell viability assays, the cytotoxic efficacy of nine jadomycin analogues (DNV, L, B, SPhG, F, W, S, T and N) in drug-sensitive and drug-resistant MCF7 breast cancer cells was evaluated. Jadomycin B, L, S and T were found to be equally toxic to drug-sensitive and drug-resistant ABCB1, ABCC1 or ABCG2-overexpressing MCF7 breast cancer cells. The inhibition of ABCB1, ABCC1 or ABCG2 efflux transporters with verapamil, MK-571 or ko143, respectively, did not significantly augment the cytotoxic effects of jadomycin DNV, L, B and S in drug-resistant MCF7 cells, suggesting that these jadomycins are poor substrates of the targeted transporter. Furthermore, all nine jadomycin analogues did not increase the intracellular accumulation of ABCB1, ABCC1 or ABCG2 probe fluorescent substrates in HEK-293 cells, indicating that these jadomycins do not inhibit the efflux function of the transporters. We conclude that jadomycins B, L and S are effective agents in the eradication of resistant breast cancer cells grown in culture, and that the ability of specific jadomycins to retain cytotoxic efficacy in resistant cells stems from their limited interactions with ABCB1, ABCC1 or ABCG2 efflux transporters.

Chemotherapy is used in the treatment of cancer. Unfortunately, drugs often fail due to multidrug resistance (MDR) caused by P-glycoprotein (P-gp1or ABCB1) and the multidrug resistance-associated protein (MRP1 or ABCC1). These proteins bind and transport drugs out of cancer cells, thereby conferring MDR. / The second chapter of this thesis addresses an unexplained phenomenon that accompanies P-gp1 expression, collaterally sensitive to verapamil. The collective results of this work demonstrated that treatment of cells that over-express P-gp1 with verapamil induces apoptosis. Furthermore, the findings show that the ATPase activity of P-gp1 was activated by verapamil. The degree of ATPase activation was proportional to the level of apoptosis and the increased demand for ATP resulted in the production of reactive oxygen species (ROS). Finally, the production of ROS led to cell death mediated by apoptosis in that experimental model system. / Chapters three and four are devoted to understanding the binding characteristics of MRP1 with two of its physiological substrates, glutathione (GSH) and leucotriene C4(LTC4). Photoreactive derivatives of these substrates were synthesised to address this objective, IAAGSH and IAALTC4. Photolabelling and transport studies showed that these derivatives have similar binding characteristics as the native compounds. In addition, photolabelling of MRP1 occurred with a high specificity with both compounds. IAAGSH and IAALTC 4 were also used to determine the locations of GSH and LTC4 binding sites. This was accomplished using MRP1-variants containing hemagglutinin (HA) epitopes at specific locations in the amino acid sequence. Through photoaffinity labelling, immunoprecipitation, and trypsin digestion, a map of binding sites for IAAGSH or IAALTC4 was obtained. Both LTC4 and GSH bound to transmembrane (TM) regions 10-11 and 16-17 which have been previously implicated in drug binding. Furthermore, novel binding sites for both substrates were discovered. IAALTC4 photolabelled a novel site within the first five TMs (TMD0) of MRP1, whereas IAAGSH labelled two cytoplasmic regions (L1 and L0). These may represent specific binding sites for LTC4 and GSH. / The work within this thesis explores some of the biochemical characteristics of Pgp1 and MRP1 that are not directly related to drug resistance and may lead to new strategies in cancer treatment.

Appendix included in back. / Errata posted on back end cover. / Bibliography: leaves 236-268. / xxii, 268, [7] leaves, [41] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / A contribution to the knowledge of multidrug resistance and its role in acute leukaemia. / Thesis (Ph.D.)--University of Adelaide, Dept. of Cytogenetics and molecular genetics, 1997

Thesis (Ph. D.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 200-242). Also available on the World Wide Web.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 6 uppsatser.

Thesis (M.Sc. (Clinical Tropical Medicine))--Mahidol University, 1999.

Thesis (M.Sc. (Clinical Tropical Medicine))--Mahidol University, 1999.