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.

Doctor of Philosophy (PhD) / The H69CIS200 cisplatin-resistant and H69OX400 oxaliplatin-resistant cell lines developed as part of this study, are novel models of low-level platinum resistance. These resistant cell lines do not have common mechanisms of platinum resistance such as increased expression of glutathione or decreased platinum accumulation. Rather, these cell lines have alterations in their cell cycle allowing them to proliferate rapidly post drug treatment in a process known as ‘regrowth resistance’. This alteration in cell cycle control has come at the expense of DNA repair capacity. The resistant cell lines show a decrease in nucleotide excision repair and homologous recombination repair, the reverse of what is normally associated with platinum resistance. The alterations in these DNA repair pathways help signal the G1/S checkpoint to allow the cell cycle to progress despite the presence of DNA damage. The decrease in DNA repair capacity has also contributed to the development of chromosomal alterations in the resistant cell lines. Similarities in chromosomal change between the two platinum resistant cell lines have been attributed to inherent vulnerabilities in the parental H69 cells rather than part of the mechanism of resistance. The H69CIS200 and H69OX400 resistant cells are cross-resistant to both cisplatin and oxaliplatin. This demonstrates that oxaliplatin does not have increased activity in low-level cisplatin-resistant cancer. Oxaliplatin resistance also developed more rapidly than cisplatin resistance suggesting that oxaliplatin may be less effective than cisplatin in the treatment of SCLC. The resistant cell lines have also become hypersensitive to taxol but show no alterations in the expression, polymerisation or morphology of tubulin. Rather, the PI3K/Akt/mTOR pathway is involved in both platinum resistance and taxol sensitivity as both are reversed with rapamycin treatment. mTOR is also phosphorylated in the resistant cell lines indicating that platinum resistance is associated with an increase in activity of this pathway. The mechanism of regrowth resistance in the platinum-resistant H69CIS200 and H69OX400 cells is a combination of activation of PI3K/Akt/mTOR signalling and alterations in control of the G1/S cell cycle checkpoint. However, more work remains to determine which factors in these pathways are governing this novel mechanism of platinum resistance.

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.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Sydney, 2007. / Title from title screen (viewed 10 June 2008). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Discipline of Medicine, Faculty of Medicine. Degree awarded 2007; thesis submitted 2006. Includes bibliographical references. Also available in print form.

Thesis (M.S.)--University of Alabama at Birmingham, 2009. / Title from first page of PDF file (viewed on June 11, 2009). Includes bibliographical references (p. 44-54).

Clinical ototoxicity - permanent hearing loss caused by medications - is estimated to affect millions of patients annually. Two classes of drug are largely to blame: platinum-based chemotherapeutics, primarily cisplatin, and aminoglycoside antibiotics. Development of methods to prevent ototoxicity depends upon an understanding of its mechanisms and may benefit from an understanding of native protective pathways of the inner ear. As the mechanisms behind cisplatin ototoxicity remain unclear, I first sought, and herein report, a refined mouse model of cisplatin ototoxicity which will allow for further in vivo investigation of cisplatin ototoxicity and potential methods for its prevention. This low-dose, multi-cycle model was found to accurately reproduce cisplatin ototoxicity as it has been described clinically and histopathologically. I then used this mouse model of cisplatin ototoxicity to investigate cisplatin pharmacokinetics in the cochlea and their role in driving cisplatin ototoxicity. Cisplatin was found to be retained within the cochlea for months following its administration. This initial finding in mice was extended to cochlear tissue samples from deceased human patients. Analysis of intra-cochlear cisplatin distribution in murine and human tissue identified the stria vascularis region of the cochlea as a promising target for intervention. With the nature of aminoglycoside ototoxicity better understood, I investigated a native inner ear protective pathway which could be leveraged to promote sensory hair cell survival. The improved hair cell survival that has previously been demonstrated as a result of heat stress was found to be mediated by cell-cell communication via extracellular vesicles. Further, hair cell protection against aminoglycosides could be reproduced through the application of exogenous, non-inner ear-derived extracellular vesicles. In sum, these data provide new insight into mechanisms of ototoxicity and details of cellular pathways which can help protect against it.

xviii, 125 p. : ill. (some col.) / Since its approval by the FDA in 1978 cisplatin (cis-diamminedichloroplatinum(II)) has revolutionized the treatment of several cancer types, particularly testicular cancer which now has a cure rate greater than 90%. Following the example set by its success, a broad range of antitumor metallodrugs is being developed. One of the most promising of these drugs, currently in Phase Two of clinical trials, is the Ru-based NAMI-A (imadozolium trans -[tetrachloro(dimethylsulfoxide)(imidazole)ruthenate(III)]) which displays low systemic toxicity and strong antimetastatic activity. The majority of anticancer metallodrugs (including NAMI-A and cisplatin) can bind to DNA, which, in many cases, is an important therapeutic target. Much effort has gone into characterizing the DNA binding properties of anticancer metallodrugs. Less study has gone into characterizing the interaction of anticancer mellodrugs with RNA even though RNA is chemically similar to DNA and plays important roles in gene expression and regulation. Focusing on the extensively studied cisplatin, Chapter I covers both what is known about anticancer metallodrug-RNA binding and the information that can be gleaned from DNA binding and drug localization studies. Chapter II provides the details of a kinetic investigation of the in vitro binding of aquated cisplatin to an RNA sequence containing an internal loop derived from the core of the spliceosome, a related RNA hairpin, and the slower reacting DNA hairpin analog. Chapter III follows in cellulo studies with cisplatin-treated S. cerevisiae that demonstrate, using ICP-MS, differences in Pt accumulation in mRNA and rRNA. The effects of cisplatin treatment on S. cerevisiae cell growth and viability were investigated using clonogenic and morphologic assays. In Chapter IV the same protocols were applied in order to investigate Ru accumulation on RNA following S. cerevisiae treatment with NAMI-A. These in cellulo experiments were followed by in vitro binding studies that utilized MALDI-MS to compare Ru interactions with RNA and DNA oligonucleotides following treatment with NAMI-A under different solution conditions, finding enhanced binding in an acidic, reducing environment like that found in tumor tissue. Chapter V pulls together the knowledge gained so far and discusses questions for future investigation. This dissertation includes both previously published and unpublished coauthored material. / Committee in charge: David Tyler, Chairperson; Victoria DeRose, Advisor; Darren Johnson, Member; Andy Berglund, Member; Alice Barkan, Outside Member

Resumo: A neuropatia induzida por drogas quimioterápicas é uma complicação no tratamento do câncer e outras doenças por ser freqüentemente dolorosa e requerer a interrupção da terapia. O antitumoral cisplatina é comumente usado contra muitas formas de câncer há aproximadamente 40 anos. Entretanto, sua aplicação é associada a muitos efeitos tóxicos, como neurotoxicidade, nefrotoxicidade, perda da audição e vômitos. Estes efeitos adversos têm levado ao desenvolvimento de agentes específicos para amenizar a toxicidade do fármaco. Alguns estudos sugerem que a administração de antioxidantes é capaz de reduzir os danos e proteger os tecidos. Dessa forma, os carotenóides são mais uma opção a ser avaliada, pois são considerados eficazes agentes antioxidantes. O urucum é uma fonte natural de corantes vermelhos e além da bixina (fração lipossolúvel do extrato), estão presentes nas suas sementes, outros carotenóides, como a norbixina, o bcaroteno, a criptoxantina, a luteína e a zeaxantina. Neste estudo, foi avaliada a genotoxicidade e a antigenotoxicidade do urucum e da bixina sobre a toxicidade induzida pelo antitumoral cisplatina em culturas de células PC12. A citotoxicidade foi determinada pelo método do MTT, a frequência de danos cromossômicos pelo Teste do Micronúcleo e a extensão de danos primários ao DNA pelo Ensaio do Cometa. O urucum e a bixina foram avaliados preliminarmente quanto a sua genotoxicidade. O urucum nas concentrações 0,2, 0,5 e 1,0 mg/mL e a bixina nas concentrações 0,05, 0,08 e 0,10 mg/mL não foram citotóxicos e nem genotóxicos às células PC12. Assim, essas concentrações foram utilizadas nos experimentos para verificar a proteção do urucum e da bixina contra os danos induzidos pela cisplatina. Embora o efeito protetor do urucum e da bixina não tenha sido evidente nos resultados obtidos pelo Ensaio do Cometa, eles se mostraram... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The neuropathy induced by chemotherapeutic drugs is a complication in the treatment of cancer and other diseases, because it is often painful and requires discontinuation of the therapy. Cisplatin has been commonly used against many forms of cancer for approximately 40 years. However, its application is associated with many toxic effects such as neurotoxicity, nephrotoxicity, hearing loss and vomiting. These adverse effects have led to the development of specific agents to lessen the toxicity of the drug. Some studies have suggested that the administration of antioxidants is able to reduce the damage and protect the tissues. Thus, the carotenoids are one more option to be evaluated, because they are considered to be effective antioxidants. Annatto is a natural source of red dyes and pigments and in addition to bixin (liposoluble fraction of the extract), other carotenoids are present in its seeds, such as norbixin, B-carotene, cryptoxanthin, lutein and zeaxanthin. In the present study, the genotoxicity and antigenotoxicity of annatto and bixin on the cisplatin induced-toxicity in PC12 cell cultures was assessed. Cytotoxicity was determined by the MTT assay, chromosomal damage by the Micronucleus test and the extent of primary damage to the DNA by the Comet assay. Annatto and bixin were first assessed with respect to their genotoxicity. Annatto concentrations of 0.2, 0.5 and 1.0 mg/ml and bixin concentrations of 0.05, 0.08 and 0.10 mg/ml were neither cytotoxic nor genotoxic to the PC12 cells. Thus, these concentrations were used in experiments to verify the protective effect of annatto and bixin against damage induced by cisplatin. Although the protective effect of annatto and bixin was not evident in the results obtained by the Comet assay, effective inhibition of the chromosomal damage (Micronucleus test) induced by cisplatin was shown. Annatto and bixin protected... (Complete abstract click electronic access below) / Orientador: Maria de Lourdes Pires Bianchi / Coorientador: Antonio Cardozo dos Santos / Banca: Alessandro de Oliveira Rios / Banca: Daisy Maria Fávero Salvadori / Banca: João Bosco Faria / Banca: Cecilia Rodrigues Silva / Doutor

Made available in DSpace on 2014-06-11T19:23:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-28Bitstream added on 2014-06-13T18:51:10Z : No. of bitstreams: 1 castro_jht_me_jabo.pdf: 401295 bytes, checksum: 5932eead976a77a78dee0fb6c3fb9169 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A correta orientação do Médico Veterinário, aos proprietários de cães, usados com finalidades reprodutivas, submetidas à quimioterapia com cisplatina, é importante na medida que este agente citostático age nas células em constante divisão, podendo ser citotóxicos para as células germinativas testiculares. O objetivo desse trabalho foi avaliar a qualidade espermática através do espermograma de cães que receberam cisplatina em diferentes momentos de análise espermática. A dose utilizada foi de 70 mg/mø, em intervalos de 21 dias, totalizando 4 infusões. Os cães foram divididos em dois grupos de 4 animais cada, sendo que um dos grupos recebeu a quimioterapia e o protocolo de diurese para proteção renal, já o grupo controle não recebeu a cisplatina, estando sujeito apenas aos fatores ambientais. Os resultados obtidos demonstraram que a cisplatina influenciou na qualidade espermática de cães, pois elevou as patologias maiores e totais acima do aceitável para cães aptos a reprodução. Portanto, infere-se que este citostático possa acarretar alterações morfofuncionais nos túbulos seminíferos e conduto epididimário. / The correct veterinary`s orientation for male dogs` owners used for reproduction goals, undergone cisplatin administration, is important because of this cistostatic act in cell with frequently proliferation, and could to cause germ cell injury. The objections of this experiment was to analysis the sperm quality through dogs` spermogram that received cisplatin`s infusions. The dose used was 70 mg/mø in 21 days periods, with 4 infusion in total. The dogs were divided in 2 groups with 4 animal each one. One of the groups received all the diuresys protocol (to protect the kidney) and the citostatic. And the other control group just didn`t receive the cisplatin infusion to know the real action of cisplatin effects without environmental stresses. The results show that cisplatin influence at the sperm quality in the dogs, because it elevated the major and total defects above that would be acceptable for competent dog to reproduct. It could deduct that cisplatin cause phisiologic alteration in the testis and epididymis.