Doctor of Philosophy(PhD) / The present study deals with the synthesis and characterization of six tri-palladium complexes code named MH3, MH4, MH5, MH6, MH7 and MH8 that contained two planaramine ligands bound to the central or each of the terminal metal ions. The activity of the compounds against human cancer cell lines: A2780, A2780cisR and A2780ZD0473R, cell uptake, levels of DNA-binding and nature of interaction with salmon sperm and pBR322 plasmid DNA have also been determined. Whereas cisplatin binds with DNA forming mainly intrastrand GG adduct that causes local bending of a DNA strand, the tri-palladium complexes are expected to bind with DNA forming a number of long-range interstrand GG adducts that would cause a global change in DNA conformation. Among the designed complexes, MH6 that has two 2-hydroxypyridine ligands bound to each of the two terminal palladium ions is found to be most active. The compound also has the highest cell uptake and Pd-DNA binding levels. In contrast, MH8 which has two 4-hydroxypyridine ligands bound to each of the two terminal palladium ions is found to be least active. The results indicate that, as applied to the terminal metal centres, 2-hydroxypyridine would be more activating than 4-hydroxypyridine perhaps because of greater protection provided to the terminal centres from coming in contact with the solvent molecules. In contrast, when bound to the central metal centre, 4-hydroxypyridine appears to play a slightly greater activating role than 2-hydroxypyridine or 3-hydroxypyridine, suggesting that non-covalent interactions such as hydrogen bonding associated with the ligand rather than its steric effect may be a more important determinant of antitumour property. The results illustrate structure-activity relationships and suggest that the tri-palladium complex containing two 2-hydroxypyridine ligands bound to each of the three metal centres or the compound that contains two 2-hydroxypyridine ligands bound to each of the two terminal metal centres and two 4-hydroxypyridine ligands bound to the central metal centre, may be much more active than any of the designed complexes.

Background: Insulin is a hormone released by the pancreatic beta cells in response to elevated levels of nutrients in the blood. Insulin triggers the uptake of glucose, fatty acids and amino acids into the liver, adipose tissue and muscles. Genes regulating insulin signalling are thus of vital importance for metabolic homeostasis and for preventing the development of diabetes. This thesis aims to identify new modifiers of insulin signalling, while carrying out functional studies of a homolog to human arsenite translocating ATPase, ASNA1. ASNA1 activates the insulin signalling pathway and promotes insulin secretion in mammalian cell lines and in Caenorhabditis elegans. A second aim is to better understand how ASNA1 and its interactors regulate sensitivity to the chemotherapeutic drug, cisplatin. Results: Regulators of insulin/IGF signalling (IIS) in C. elegans were identified based on the Larval arrest arrest aspect of the asna-1 depletion phenotype. Sixty-five genes were selected by virtue of their predicted interaction with ASNA-1 and screened for asna-1-like larval arrest upon inactivation of the genes . mrps-2, mrps-10, mrpl-43 encoding mitochondrial ribosomal protein subunits, and enpl-1 encoding an ER chaperone, GRP94 homolog were identified as the genes which when inactivated caused larval arrest without any associated feeding defects. IIS was weaker and insulin secretion was defective in these knockdown animals. ENPL-1 and ASNA-1 proteins interacted with one another both ex vivo and in vitro. ASNA-1 protein and mRNA level swere greatly reduced in enpl-1 mutants and enpl-1(-);asna-1(-) double-mutant worms displayed synthetic lethality. Overexpression of the insulins INS-4 and DAF-28 caused partial rescue of the germline phenotype of enpl-1 mutants, indicating that the phenotype of enpl-1 mutants was due at least in part to insufficient insulin levels. Studies of enpl-1 mutants also helped to understand the role of asna-1 in cisplatin sensitivity. The unfolded protein response (UPR) was induced in asna-1 and enpl-1 knockdown animals. enpl-1 mutants displayed higher sensitivity to cisplatin, when compared to asna-1 mutants and this correlated to higher UPR in enpl-1 knockdown animals. Pharmacological induction of the UPR in intrinsically cisplatin resistant wildtype worms also resulted in increased cisplatin sensitivity. This suggests that manipulation of ENPL-1 levels or of the UPR could enhance the anti-tumoral effects of cisplatin based cancer therapy. With a yeast two hybridscreen 27 putative physical interactors of ASNA-1 were identified. Amongst these candidate swas smn-1, which encodes survival of motor neuron protein homolog. RNAi knockdown of smn-1 caused a larval arrest phenotype similar to asna-1 depleted animals and smn-1 positively regulated IIS, like asna-1. Defects in IIS may be at the level of insulin release because neuropeptide secretion was impaired upon smn-1 knockdown. Further in vitro binding studies showed that SMN-1 and ASNA-1 interacted and inactivation of smn-1 in asna-1 mutants resulted in decreased viability. This implies that SMN-1 is another modifier of ASNA-1 and also a new component in IIS. Conclusion: With a directed RNAi screen and a yeast two hybrid screen several interactors of ASNA-1 that are also IIS modifiers were identified. ENPL-1 and SMN-1 are both involved in insulin release. We also found that induction of the UPR in enpl-1 and asna-1 mutants is a possible mechanism for increased sensitivity to cisplatin.

Understanding the specific mechanisms regulating chemotherapeutic drug anti-cancer activities will uncover novel strategies to enhance the efficacy of these drugs in clinical settings. Activating Transcription Factor 3 (ATF3) is a stress inducible gene whose expression has been associated with survival outcomes in cancer models. This study characterizes the chemotherapeutic drugs, cisplatin and Histone Deacetylase Inhibitor (HDACi), M344 as novel inducers of ATF3 expression. Cisplatin is a DNA damaging agent widely used in various tumour types including lung, head and neck, and ovarian carcinomas. The HDAC inhibitor, SAHA, has recently been approved as a single agent in the treatment of subcutaneous T-cell lymphoma and HDACis themselves show potential for synergistic anti-cancer effects when used in combination with established chemotherapeutic drugs, including cisplatin. This study evaluates the mechanisms by which cisplatin and HDACi induce ATF3, as well as the role ATF3 plays as a mediator of cisplatin-induced cytotoxicity and the enhanced cytotoxicity between HDACi and cisplatin in combination. In this study, we demonstrate that cytotoxic doses of cisplatin and carboplatin consistently induced ATF3 expression in a panel of human tumour derived cell lines. Characterization of this induction revealed a p53, BRCA1, and integrated stress response (ISR) independent mechanism, all previously implicated in stress mediated ATF3 induction. Analysis of MAPKinase pathway involvement in ATF3 induction by cisplatin revealed a MAPKinase dependent mechanism. Cisplatin treatment, in combination with specific inhibitors to each MAPKinase pathway (JNK, ERK and p38) resulted in decreased ATF3 induction at the protein level. MAPKinase pathway inhibition led to decreased ATF3 mRNA expression and a reduction in the cytotoxic effects of cisplatin as measured by MTT cell viability assay. In A549 lung carcinoma cells, targeting ATF3 with specific shRNAs also attenuated the cytotoxic effects of cisplatin. Similarly, ATF3 -/- MEFs were shown to be less sensitive to cisplatin induced cytotoxicity as compared with ATF3+/+ MEFs. Taken together, we identified cisplatin as a MAPKinase pathway dependent inducer of ATF3 whose expression regulates in part cisplatin’s cytotoxic effects. Furthermore, we demonstrated that the HDAC inhibitor M344 was also an inducer of ATF3 expression at the protein and mRNA level in the same human derived cancer cell lines. Combination treatment with M344 and cisplatin lead to increased induction of ATF3 compared with cisplatin alone. Utilizing the MTT cell viability assay, M344 treatment was also shown to enhance the cytotoxic effects of cisplatin in these cancer cell lines. Unlike cisplatin, the mechanism of ATF3 induction by M344 was found to be independent of MAPKinase pathways. Utilizing ATF4 heterozygote (+/-) and knock out (-/-) mouse embryonic fibroblast (MEF) M334 induction of ATF3 was shown to depend on the presence of ATF4, a known regulator of ATF3 expression as part of the ISR pathway. HDACi treatment did not affect the level of histone acetylation associated with the ATF3 promoter as determined through Chromatin immunoprecipitation (ChIP) analysis, suggesting that ATF3 induction was not a direct effect of HDACi mediated histone acetylation. We also demonstrated that ATF3 regulates the enhanced cytotoxicity of M344 in combination with cisplatin as evidenced by attenuation of cytotoxicity in shRNAs targeting ATF3 expressing cells. This study identifies the pro-apoptotic factor, ATF3 as a novel target of M344, as well as a mediator of the co-operative effects of cisplatin and M344 induced tumour cell cytotoxicity.

Platinum-based chemotherapies are effective agents in the treatment of a wide variety of human cancers. However, patients with recurrent disease can become resistant to platinum-based chemotherapy, leading to low overall survival rates. Activating transcription factor 3 (ATF3) is a stress-inducible gene that is a regulator of cisplatin-induced cytotoxicity. ATF3 protein expression was upregulated after cytotoxic doses of cisplatin treatment in a panel of cell lines. A chromatin immunoprecipitation assay showed that upon treatment with cisplatin, ATF3 directly bound to the CHOP gene promoter and this correlated with an increase in CHOP protein expression. In a 1200 compound library screen performed on cancer cell lines, disulfiram, a dithiocarbamate drug, was identified as an enhancer of the cytotoxic effects of cisplatin. This increased cytotoxic action was likely due to disulfiram and cisplatin’s ability to induce ATF3 independently through two separate mechanisms, namely the MAPK and integrated stress pathways. Furthermore, ATF3 protein and mRNA levels were variable amongst human ovarian and lung cancer tissues, suggesting the potential for basal expression of ATF3 to be predictive of cisplatin treatment response. Thus, understanding ATF3’s role in cisplatin-induced cytotoxicity will lead to novel therapeutic approaches that could improve this drug’s efficacy.

No description available.

Understanding the specific mechanisms regulating chemotherapeutic drug anti-cancer activities will uncover novel strategies to enhance the efficacy of these drugs in clinical settings. Activating Transcription Factor 3 (ATF3) is a stress inducible gene whose expression has been associated with survival outcomes in cancer models. This study characterizes the chemotherapeutic drugs, cisplatin and Histone Deacetylase Inhibitor (HDACi), M344 as novel inducers of ATF3 expression. Cisplatin is a DNA damaging agent widely used in various tumour types including lung, head and neck, and ovarian carcinomas. The HDAC inhibitor, SAHA, has recently been approved as a single agent in the treatment of subcutaneous T-cell lymphoma and HDACis themselves show potential for synergistic anti-cancer effects when used in combination with established chemotherapeutic drugs, including cisplatin. This study evaluates the mechanisms by which cisplatin and HDACi induce ATF3, as well as the role ATF3 plays as a mediator of cisplatin-induced cytotoxicity and the enhanced cytotoxicity between HDACi and cisplatin in combination. In this study, we demonstrate that cytotoxic doses of cisplatin and carboplatin consistently induced ATF3 expression in a panel of human tumour derived cell lines. Characterization of this induction revealed a p53, BRCA1, and integrated stress response (ISR) independent mechanism, all previously implicated in stress mediated ATF3 induction. Analysis of MAPKinase pathway involvement in ATF3 induction by cisplatin revealed a MAPKinase dependent mechanism. Cisplatin treatment, in combination with specific inhibitors to each MAPKinase pathway (JNK, ERK and p38) resulted in decreased ATF3 induction at the protein level. MAPKinase pathway inhibition led to decreased ATF3 mRNA expression and a reduction in the cytotoxic effects of cisplatin as measured by MTT cell viability assay. In A549 lung carcinoma cells, targeting ATF3 with specific shRNAs also attenuated the cytotoxic effects of cisplatin. Similarly, ATF3 -/- MEFs were shown to be less sensitive to cisplatin induced cytotoxicity as compared with ATF3+/+ MEFs. Taken together, we identified cisplatin as a MAPKinase pathway dependent inducer of ATF3 whose expression regulates in part cisplatin’s cytotoxic effects. Furthermore, we demonstrated that the HDAC inhibitor M344 was also an inducer of ATF3 expression at the protein and mRNA level in the same human derived cancer cell lines. Combination treatment with M344 and cisplatin lead to increased induction of ATF3 compared with cisplatin alone. Utilizing the MTT cell viability assay, M344 treatment was also shown to enhance the cytotoxic effects of cisplatin in these cancer cell lines. Unlike cisplatin, the mechanism of ATF3 induction by M344 was found to be independent of MAPKinase pathways. Utilizing ATF4 heterozygote (+/-) and knock out (-/-) mouse embryonic fibroblast (MEF) M334 induction of ATF3 was shown to depend on the presence of ATF4, a known regulator of ATF3 expression as part of the ISR pathway. HDACi treatment did not affect the level of histone acetylation associated with the ATF3 promoter as determined through Chromatin immunoprecipitation (ChIP) analysis, suggesting that ATF3 induction was not a direct effect of HDACi mediated histone acetylation. We also demonstrated that ATF3 regulates the enhanced cytotoxicity of M344 in combination with cisplatin as evidenced by attenuation of cytotoxicity in shRNAs targeting ATF3 expressing cells. This study identifies the pro-apoptotic factor, ATF3 as a novel target of M344, as well as a mediator of the co-operative effects of cisplatin and M344 induced tumour cell cytotoxicity.

Platinum-based chemotherapies are effective agents in the treatment of a wide variety of human cancers. However, patients with recurrent disease can become resistant to platinum-based chemotherapy, leading to low overall survival rates. Activating transcription factor 3 (ATF3) is a stress-inducible gene that is a regulator of cisplatin-induced cytotoxicity. ATF3 protein expression was upregulated after cytotoxic doses of cisplatin treatment in a panel of cell lines. A chromatin immunoprecipitation assay showed that upon treatment with cisplatin, ATF3 directly bound to the CHOP gene promoter and this correlated with an increase in CHOP protein expression. In a 1200 compound library screen performed on cancer cell lines, disulfiram, a dithiocarbamate drug, was identified as an enhancer of the cytotoxic effects of cisplatin. This increased cytotoxic action was likely due to disulfiram and cisplatin’s ability to induce ATF3 independently through two separate mechanisms, namely the MAPK and integrated stress pathways. Furthermore, ATF3 protein and mRNA levels were variable amongst human ovarian and lung cancer tissues, suggesting the potential for basal expression of ATF3 to be predictive of cisplatin treatment response. Thus, understanding ATF3’s role in cisplatin-induced cytotoxicity will lead to novel therapeutic approaches that could improve this drug’s efficacy.

Cisplatin (CisPt) is an important drug that is used against various cancers, including testicular, ovarian, lung, head, and neck cancer. However, its effects are limited by cellular resistance. The resistance is believed to be multifactorial, and may be mediated to varying degree by multiple systems in cells, one of the proposed systems being the copper (Cu) transporting system. The Cu-importer Ctr1 has proven importance for cellular sensitivity to CisPt by regulating its influx, while the Golgi-localized Cu-ATP:ases ATP7A/B can putatively mediate CisPt efflux and/or drug sequestration. Atox1 is a small Cu-chaperone that normally transfers Cu between Ctr1 and ATP7A/B, prior to delivery of Cu to the proteins in the secretory pathway. Since Ctr1 and ATP7A/B are reportedly involved in CisPt-resistance, CisPt interaction with Atox1 was the focus of the project this thesis is based upon.   Using a variety of techniques, Atox1 was found to bind CisPt, also simultaneously with Cu. The Atox1-CisPt complexes were further probed using selected mutants in studies demonstrating that only the two cysteines (Cys12 and Cys15) in the Cu-binding site of Atox1 are essential for CisPt interactions. A proposed Atox1 di-metal complex containing both Cu and CisPt was found to be monomeric, and no loss of Cu was observed. In vitro experiments demonstrated that CisPt could also bind to metal-binding domain 4 of ATP7B (WD4), and that the drug could be transferred from Atox1 to the domain. These findings indicated that Atox1 may transfer CisPt to ATP7A/B in vivo, utilizing the same transport pathway as Cu. However, the CisPt-bound Atox1 complexes were not stable over time; upon incubation, protein unfolding and aggregation were observed. Thus, in vivo, Atox1 might alternatively be a dead-end sink for CisPt.   The effects of the ligands around the Pt-center of Pt-based anticancer drugs and drug derivatives on Atox1 binding and unfolding were also investigated. The ligands’ chemistry and geometry were shown to dictate the extent and rate of the Pt-based substances interactions with Atox1. Finally, the occurrence of Atox1-CisPt interactions in a biological environment was demonstrated by developing and applying an antibody-based method allowing analysis of metals associated with Atox1 extracted from CisPt-treated cells.   The findings presented in this thesis show that CisPt binds to Atox1 and WD4, also simultaneously with Cu, in vitro. The results support the hypothesis that Cu-transporting proteins can mediate cellular resistance to CisPt in vivo, and provide a deeper chemical understanding of the interactions between the proteins and the drug.

Nasopharyngeal carcinomas (NPC) is highly induced by Epstein-Barr virus (EBV). EBV infection encoded latent membrane protein 1 (LMP1) is expressed in latent stage II and III of EBV infection nasopharyngeal cells. LMP1 was reported to be associated with increased tumorigenesis, through the activation of nuclear factor-£eB (NF-£eB). In this study, LMP1 overexpressing NPC cell line (TW04) was used for assays of cell proliferation, apoptosis and migration under drug (Sodium Salicylate, Cisplatin and 5-Fluorouracil) treatment. Sodium Salicylate is one of non-steroidal anti-inflammatory (NSAID) drug, which inhibits the downstream of NF-£eB pathway, e.g., COX-1. Cisplatin and 5-Fluorouracil are traditional chemotherapy durgs used in many cancers. Our result shows that overexpression of LMP1 affects cell proliferation, apoptosis , invasion and migration ability. It indicates that LMP1-overexpression is an important marker for NPC therapy.

The present study explored the effect of three anti-tumor drugs (cisplatin, fluorouracil, and temozolomide) on viability and cytosolic free Ca2+ concentrations ([Ca2+]i) in OC2 human oral cancer cells. The effect of cisplatin related mitogen-activated protein kinases (MAPKs) phosphorylation was also examined. Cisplatin at concentration of 25-150 £gM decreased viability in a concentration-dependent manner, and so did fluorouracil (50-1000 £gM) and temozolomide (50-600 £gM). The three anti-tumor drugs all failed to induce a [Ca2+]i increase; thus it seemed that these drugs induced cell death via Ca2+-independent pathways. Immunoblotting showed that OC2 cells have background phospho-ERK, phospho-JNK and phospho-p38 MAPKs. It was found that cisplatin influenced the phosphorylation of ERK, JNK and p38 MAPKs at different time points.