Dissertations / Theses on the topic 'Breast cancer; Novel anticancer agents'

Thesis (M.S.)--University of Toledo, 2008.<br>Typescript. "Submitted as partial fulfillments of the requirements for The Master of Science in Pharmaceutical Sciences in Pharmacology and Toxicology." "A thesis entitled"--at head of title. Bibliography: leaves 47-49.

Many studies have reported the anti-cancer effects of n-3 long chain polyunsaturated fatty acids (n-3 LCPUFAs). In this study, the anticancer effects of n-3 LCPUFAs and their derivatives, n-3 n-acyl ethanolamides (n-3 NAEs) were investigated in MCF-7 and MDA-MB-231 breast cancer cell lines. The focus was on their effects on cell proliferation, the role of CB1 and CB2 receptors, the effects of their main metabolising enzyme, FAAH, and which molecular signalling pathways are involved. In addition, the effects on the redox system, particularly in the modulation of redox genes, proteins and enzymes, the role of epigenetic regulation, and the effects on cell invasion and migration were also investigated. The results showed that n-3 NAEs were more potent than their parent LCPUFAs at reducing cell viability while the effects of both n-3 LCPUFAs and n-3 NAEs appear to be CB receptor mediated. In addition, MAPK pathways were also affected to varying extents following treatment, in particular p38 and JNK. Furthermore, global methylation, antioxidant gene expression, migration and invasion were in general all modulated by treatment. However, the effects observed were found to be both treatment- and cell- type dependent. Nevertheless, these results confirm that n-3 LCPUFAs and n-3 NAEs inhibit breast cancer cell growth, and modulate important cancer related pathways albeit by different mechanisms. This suggests that dietary intervention with cheap, safe, readily available fatty acids could be introduced to breast cancer patients to enhance treatment.

Philosophiae Doctor - PhD<br>Breast cancer is one of the most common cancers among women in South Africa and the second leading cause of cancer death after lung cancer. According to the American Cancer Society 2015, women have a 12% chance of developing invasive breast cancer and a 3% chance of dying from it. Despite the wide variety of breast cancers e.g. lobular carcinoma in situ (LCIS) and ductal carcinoma in situ (DCIS), many share the same etiology and target tissue. Estrogen related carcinogenesis with regard to breast cancer typically results from the activation of distinct signalling pathways. These pathways are not mutually exclusive and are often constituted by receptor mediated stimulation of cell proliferation caused by specific transcriptional gene activation, reactive oxygen species (ROS) formation causing DNA damage and consequently mutations. The molecular pathways that cause drug resistance are not fully understood and the search continues to find novel targets for treatment. The effects of non-toxic triterpenes, oleanolic acid and ursolic acid and the role of autophagy and apoptosis as mechanisms to overcome drug resistance in breast cancer were studied in vitro in MCF-7 breast cancer cells and MCF10A breast cells. In this study the first aim was to establish the influence of OA and UA on cell growth and to see if opposing proliferation patterns could observed between the presumably ERɑ negative (ERɑ/ß -/+) MCF-10A and ERɑ positive (ERɑ/ß +/+) MCF-7 cells. This was followed by morphology studies to establish the possible presence of cytotoxicity and examination of molecular pathways contributing to the anti-cancerous properties of UA and OA and their validity as therapeutic agents. The MCF-7 breast cancer cell line and the immortalized normal mammary cell line, MCF-10A were treated with different concentrations of UA and OA for 6hrs, 12hrs, 24hrs, 48hrs, and 72hrs respectively. Cell morphology was studied in hematoxylin and eosin as well as Hoechst and acridine orange stained cells and viability was measured using crystal violet staining. Molecular techniques employed included the Tali® Apoptosis - and the cellROX assays, flow cytometry and western blotting. Morphological, viability and apoptotic studies have shown that at their lowest concentration, both UA and OA have anti-proliferative and apoptotic effects on MCF-7 and to a lesser extent on MCF-10A. Flow cytometric analysis of treated cells has demonstrated cell arrest in the S- and G2/M phase. The MCF-7 and MCF-10A cells growth inhibition effect may be due to increased autophagy and apoptosis as an alternative to decreased proliferation in MCF-7 cells. This possibility should be evaluated in further studies. The results showed that UA was more effective OA in decreasing cell numbers and it may be applied as treatment for breast cancer. Our observation has shown the treatment with OA and UA increased cell death in MCF-7 cells.The opposing proliferation patterns observed between the presumably ERɑ negative (ERɑ/ß -/+) MCF-10A and ERɑ positive (ERɑ/ß +/+) MCF-7 cells could possibly be ascribed to ERß forming homodimers that may facilitate proliferation, whereas ERɑ/ß heterodimers (expressed in 59% of breast cancers) are frequently associated with the ERɑ antagonising actions of ERß. The results indicate a trend towards biphasic and anti- proliferative effects of the reactants in breast cancer cells which may contribute towards the development of anti- cancer therapies. However, further work is must be done to identify the OA and UA mechanism(s) responsible for anticancer activity.<br>Libyan Embassy

Hepatocellular carcinoma accounts for 85% of primary liver cancer that are usually characterised by defective or ineffective apoptosis which is considered to be the main cause of cancer progression. In this study, Cytochrome-C which is a pro-apoptotic protein is combined with hybrid (iron oxide-gold) nanoparticles and triggers mitochondrial downstream apoptosis pathway in the tumour cells. The nanoparticle complex enables delivery of this difficult protein through the cell membrane. In this research, five different anticancer drugs (doxorubicin, oxaliplatin, paclitaxel, vincristine and vinblastine) were used against liver cancer and U937 cell lines to assess their IC50 values alone and to check their toxicity after their co-administration with cytochrome C hybrid formulation. These combinations resulted in an increase in the cytotoxicity of the used chemotherapeutic drugs and remarkable decrease in the amount needed to kill hepatic cancer cells. For that reason, Iron-gold hybrid nanoparticles offer a promising tool for cytochrome-c delivery into tumour cells and enhance the specific targeting of therapeutic particles to their site of action. The preliminary results reflected the increasing killing abilities of chemotherapeutic therapies when co-administered with cytochrome C hybrid formulation by targeting the natural killing mechanism inside the cells and activating its pathways. Subsequent to these results, further work was done in formulation of one platform therapeutic device with Polymeric amphiphiles hydrophilic poly(allylamine) polymer (PAA) grafting with 5-(4-chlorophenyl)-1,3,4-oxadiazole-2-thiol(oxadiazole, Ox). Paclitaxel (PTX) was selected as a hydrophobic drug model to check its water solubility behaviour after loading into PAA-HNP-C platform. These new devices showed a significant increase in drug uptake level and increase in PTX cytotoxicity against liver cancer cell lines. The data from this work showed a significant increase in the apoptosis activities of combining treatment anticancer agents (doxorubicin, paclitaxel, oxaliplatin, vinblastine and vincristine) and the hybrid formulation of the cytochrome-C within the liver cell lines, which leads to cellular death. Therefore, this combined method may give promise step for the future of liver cancer treatment regimes. In addition to, formulating the HNP-CYT-C and PTX into as active single platform for increasing the PTX cytotoxicity. More laboratory investigation is needed to check the activity of this formulation as a preparing step to further in vivo studies.

The Tyrrell group has, in recent years, developed a strong interest in the chemical synthesis and biological activity of the hop bitter acids lupulone 1, humulone 2 and their naturally occurring congeners, 1 a-d and 2a-d. Previous work focused on investigating the chemistry of the a-acid humulone 2, demonstrating the propensity of this compound to have a cytotoxic effect against the SK-MES lung cancer cell line and the MCF-7 breast cancer cell line. This thesis documents the recent research concerning the synthesis, the anticancer and antibacterial activity of the ß-acid lupulone 1, its naturally occurring congeners 1 a-d and further, novel, non-naturally occurring compounds. We resumed the group's previous collaboration with the Golston and Pirianov research group at St Georges, where the anticancer studies were performed. The synthesis centred on a Friedel-Crafts acylation of phloroglucinol 32, followed by a C-trialkenylation reaction between the acylphloroglucinol and an allyl bromide. We conducted experiments that focused upon the choice of base and solvent for the trialkenylation reaction, concluding that liquid ammonia as both the base and solvent offered the most efficient route to the ß-acids. It was shown that aliphatic allylic bromides lend themselves to the' reaction, . although some derivatives require purification by column chromatography in addition to recrystallisation. In contrast, aromatic allyl bromides did not participate in the alkenylation reaction. We further investigated an alternate G-alkenylation reaction involving the di¬lithiation of 1,3,5-trimethoxybenzene. We discovered that by including copper (I) iodide in the reaction, prenyl bromide 23 and allyl bromide 41 could be successfully coupled. VVe also discovered that our 2nd generation ß-acids 42a-g could participate in a ring-closing metathesis reaction, forming novel spirocyclic compounds 50a-b. Our antibacterial studies showed that ß-acids are effective against Gram-positive bacteria, but not Gram-negative bacteria in accordance with published observations. We took our investigations further and found that ß-acids are effective against mutlidrug-resistant 'Staphylococcus aureus', even where the commercially available ciprofloxacin 67 was not.

SIRT1 and SIRT2 are two NAD⁺-dependent deacetylases which negatively modulate the activity of p53, a protein which is involved in cell cycle arrest, senescence and apoptosis following genotoxic stress. Part I of the thesis describes the exploration of the chemical space around a reported unselective and modest inhibitor of SIRT1 and SIRT2 with the aim of improving the selectivity and potency of the inhibitor against the two isoforms. Particular emphasis is placed upon understanding the mode of binding of the novel analogues within the active site of the enzymes. Chapter 1 reviews the physiological roles of class III NAD⁺-dependent deacetylases, also known as sirtuins. In particular, the application of SIRT1 and SIRT2 inhibitors as potential anticancer agents is described. Amongst these, only cambinol and the tenovins showed in vivo activity in a mouse xenograft model. Previously only one analogue of cambinol had been reported in the literature. Chapter 2 describes the development of a small collection of novel cambinol analogues (First Generation Studies). The role played by different substituents at the phenyl group and at the N-1 of the thiouracil core is discussed. Along with the synthesis and structure activity relationship (SAR) associated with the core structure, in-cell experiments intended to confirm the activity of the most active compounds are reported. Chapter 3 provides a rationalisation for the SAR discussed in Chapter 2. Based on computational molecular modelling studies (GOLD), the activity of the most potent and selective SIRT2 inhibitors is explained. Two series of novel cambinol analogues were designed (Second and Third Generation Analogues) in order to assess further the proposed binding mode. Chapter 4 focuses on the development of the “Second Generation” analogues, characterised by the presence of lipophilic substituents at the sulfur atom and at the N-3 position of the thiouracil core. The synthesis, biological evaluation and SAR are discussed in detail. Chapter 5 reports the development of the “Third Generation” analogues, characterised by either a benzyl group or para-alkoxy-substituted benzyl group at the N-1 position of cambinol. Once again, the synthesis, biological evaluation and SAR data are presented. An improved understanding of the mode of binding of the novel compounds is proposed based on molecular dynamics (MD) studies. Indole-based alkaloids, such as Vincristine and Vinblastine, are well known for their anticancer activity. Recently, the anticancer activity of members of the calycanthaceous family of alkaloids has been discovered. Part II of the thesis focuses on model studies aimed at developing the total synthesis of one of these compounds, perophoramidine. Chapter 7 provides an overview of the calycanthaceous alkaloid family of natural products, including their biological properties. The structural features of perophoramidine, along with the previously reported synthetic studies are outlined. Chapter 8 describes the synthesis of an advanced intermediate in the total synthesis of dehaloperophoramidine, a structural analogue of perophoramidine Problems encountered, optimisation studies and the synthesis of a re-designed intermediate are also reported in this chapter.

Thesis (Ph. D.)--Ohio State University, 2003.<br>Title from first page of PDF file. Document formatted into pages; contains xxiii, 249 p. : ill. Advisor: Robert W. Brueggemeier, College of Pharmacy. Includes bibliographical references (p. 234-249).

La plupart des molécules utilisées en chimiothérapie conventionnelle, bien qu’ayant des cibles moléculaires différentes, induisent dans la majorité des cas une mort cellulaire par apoptose. Or, de plus en plus de chimiorésistances se rencontrent au niveau des cellules cancéreuses vis-à-vis de ce type de molécules. Face à cette situation il devient urgent de trouver des molécules ayant des mécanismes d’action différents et capables de court-circuiter spécifiquement les mécanismes de résistance des cellules cancéreuses. <p>La stratégie mise en place lors de ce travail a été de partir d’une molécule naturelle issue d’un extrait de la racine de Glycyrrhiza glabra qui présentait déjà une activité anti tumorale marquée. L’intérêt du travail a été de dériver l’acide 18β-glycyrrhétinique de manière originale afin de potentialiser son effet anticancéreux, notamment vis-à-vis de huit lignées cellulaires présentant des résistances plus ou moins marquées aux stimuli pro-apoptotiques. Ainsi après avoir caractérisé la pureté et la stabilité de cette série de nouvelles molécules, nous avons retenu les dérivés les plus intéressants en termes d’inhibition in vitro de la prolifération cellulaire. Sur base de ce premier choix, nous avons investigué des cibles spécifiques décrites dans la littérature pour les hémidérivés de l’acide 18β-glycyrrhétinique :le protéasome 26S et le récepteur nucléaire PPARγ. Cette étude nous a permis de retenir un dérivé en particulier capable d’inhiber à 50% les trois sites catalytiques du protéasome sans toutefois inhiber PPARγ :le N-(2-{3-[3,5-bis(trifluoromethyl)phenyl]ureido}ethyl)-glycyrrhetinamide (6b). Nous avons ensuite évalué ce composé sur un ensemble de 333 kinases afin de déterminer un profil antitumoral plus large pour ce type de molécule. <p>Le profil pharmacologique in vitro de ce dérivé de l’acide 18β-glycyrrhétinique nous a amenés à étudier son comportement in vivo chez la souris saine. A cette fin, une étude de préformulation nous a permis de définir une formulation galénique optimale pour ce composé, la nanoémulsion qui a servi à déterminer une dose maximale tolérée (indice DMT) par la souris saine. Nous avons ensuite travaillé à une dose non toxique pour déterminer le profil pharmacocinétique plasmatique chez la souris saine, par voie d’administration intraveineuse et par voie orale. <p>Les conclusions de cette étude nous montrent que le dérivé de l’acide 18β-glycyrrhétinique que nous avons mis au point présente de remarquables caractéristiques pharmacologiques in vitro et un comportement in vivo proche de la molécule naturelle. Des études d’activité in vivo devraient débuter prochainement.<p><br>Doctorat en Sciences biomédicales et pharmaceutiques<br>info:eu-repo/semantics/nonPublished

Methotrexate (MTX) is an antimetabolite anticancer agent that is used in treatment of multiple cancers, such as acute lymphoblastic leukaemia and osteosarcoma. A lack of selective tumour toxicity is one of the major problems associated with MTX chemotherapy, especially when given at high doses, as in high dose MTX (HDMTX) therapy. MTX causes various toxicity problems including life-threatening nephrotoxicity, haematological toxicity and neurotoxicity. Overcoming this toxicity is of great importance and has been attempted in various ways, not least via the design of prodrugs. The concept of tumour protease, and specifically matrix metalloproteinase (MMP), activated prodrugs was the focus of the work described in this thesis. This concept relies upon attachment of an MMP-sensitive peptide sequence to a specific site in a drug structure, so as to inactive it. The activity of the parent drug is restored once it is activated by the MMPs in the tumour microenvironment. In this work, different MMP-sensitive peptide sequences linked to MTX were synthesised, resulting in 63 MTX prodrugs. The MMP-mediated activation of these conjugates in tumour tissues (specifically HT1080 homogenates) ex vivo was assessed and the results were compared to the activation of these conjugates in various normal tissues specifically liver, kidney and lung. Specific criteria were established for the selection of promising conjugates for more detailed study. From 7 promising compounds, compound 75 was identified as the lead prodrug, demonstrating selective MMP activation, as indicated by inhibition of its activation by broad spectrum MMP inhibitor ilomastat. The pharmacokinetics of compound 75 was studied in tumour (HT1080) xenograft-bearing mice and the results were compared to those obtained from administration of equimolar doses of conventional MTX. Compound 75 led to enhanced tumour concentrations of MTX, with reduced exposure to normal tissues in vivo compared to conventional MTX therapy. Furthermore, the efficacy of equimolar doses of compound 75 and directly dosed MTX in reduction of HT1080 volume were compared. Superior antitumour activity was observed with compound 75 compared to MTX treatment. Compound 75 is the first example of an MMP-activated prodrug to be reported with enhanced therapeutic index, as evidenced by a full in vivo pharmacokinetic analysis and normal tissue metabolism data. The data presented in thesis support the concept of MMP-activated prodrug development, and form a strong foundation upon which to develop a clinicallyuseful MTX prodrug, with the potential to enhance efficacy and reduce toxicity to the patient.

Nanotechnology has recently emerged as a strong contributor toward research efforts to develop targeted systems of drug delivery in cancer therapy. Our work investigates the therapeutic potential of Targeted Charge-Reversal Nanoparticles (TCRNs), a novel nanoparticle with in vitro evidence of nuclear drug delivery. Using M12 prostate cancer cells, MDA-MB-231 breast cancer cells, and modified derivatives of these cell lines, we investigated the ability of Folic Acid-tagged TCRNs to deliver Nile Red and Dimethyl Indole Redfluorescent (DiR) fluorescent dyes to the nucleus of cells using confocal microscopy and in vivo biphontonic imaging using Xenogen® Technology. Confocal imaging with the SCP28 derivative of MDA-MB-231 cells shows nuclear association of the TCRNs over time, although specific nuclear deposition was unclear. Biophotonic imaging with M12 and SCP28 xenograft tumors in athymic nude mice shows retention of TCRNs in animals out to 7 days with minimal localization of TCRNs to tumor tissues. Our findings suggest that further characterization and manipulation of both the cells and the nanoparticle is necessary in order to make definitive claims regarding the TCRN’s ability to deliver fluorescent dyes, and eventually therapeutic compounds, to the nucleus of cells.

Le laboratoire a récemment identifié la protéine Damaged-DNA-Binding 2 (DDB2), connue à l’origine pour son rôle dans la réparation de l’ADN comme une protéine impliquée dans la tumorigenèse mammaire. En effet, nous avons montré son rôle dans la croissance et la progression des tumeurs mammaires via la régulation transcriptionnelle de gènes cibles. Dans ce travail, nous avons montré que la surexpression de DDB2 augmente la sensibilité des cellules tumorales MDA-MB231 et SKBr3 traitées à la doxorubicine et au 5-fluorouracile (5-FU). Inversement, l’inhibition de l’expression de DDB2 dans les cellules T47D qui l’expriment naturellement s’accompagne d’une baisse de la sensibilité à ces agents anticancéreux. Nos résultats montrent que la sensibilité des cellules au 5-FU mais pas à la doxorubicine, lorsque DDB2 est surexprimée, dépend en partie du contrôle négatif qu’exerce cette dernière sur l’activité de NF-κB, en régulant positivement l’expression d’IκBα. Enfin, la recherche d’autres gènes cibles de DDB2, impliqués dans l’apoptose, nous a conduits à celui codant le facteur anti-apoptotique Bcl-2. DDB2 agit négativement sur l’expression de Bcl-2 en interagissant avec une région de l’ADN localisée dans le promoteur P2 du gène correspondant. De part, son rôle anti-apoptotique, la régulation de son expression pourrait bien être à l’origine de la sensibilité aux agents anticancéreux induite par DDB2. L’ensemble de ces résultats met donc en évidence l’intérêt clinique de DDB2 comme marqueur prédictif de la réponse aux agents anticancéreux, et devrait contribuer à une meilleure compréhension des mécanismes impliqués dans l’échappement des cellules tumorales aux thérapies<br>The laboratory has recently identified the Damaged-DNA Binding 2 protein (DDB2), a protein involved in DNA repair, as an important actor in breast tumorigenesis. Our laboratory has shown that DDB2 is involved in breast tumor growth and progression through the transcriptional regulation of target genes. Thus, the first aim of this work was to study the role of DDB2 and its target genes in the response of breast cancer cells to anticancer drugs. We showed that DDB2 overexpressed in breast cancer cell lines, such as MDA-MB231 and SKBr3, increased the cells sensitivity to apoptosis induced by doxorubicin and 5-Fluorouracil (5-FU). Conversely, the inhibition of DDB2 expression in T47D cells, which express endogenously this protein, decreased cell sensitivity to anticancer agents. Our results showed that cell sensitivity induced by DDB2 expression to 5-FU but not doxorubicin depended on its ability to repress NF-κB activity via the regulation of IκBα expression. At last, the search of potential DDB2 target genes implicated in apoptosis has led us to identify the anti-apoptotic factor Bcl-2. We showed the ability of DDB2 to downregulate Bcl-2 expression via its interaction with DNA region located in P2 promoter of the corresponding gene. Results suggest that Bcl-2 dowregulation by DDB2 could be a major event that explains the enhanced sensitivity of cancer cells to therapeutic agents. Altogether, these data highlight the clinical interest of DDB2, as a predictive marker of the response to anticancer agents. A better understanding of its mode of action will contribute to improve therapeutic treatments and avoid their failure in resistant patients

Tese de doutoramento em Biociências, na especialidade de Bioquímica, apresentada à Faculdade de Ciências e Tecnologia da Universidade de Coimbra<br>Despite all efforts, cancer is still a growing health problem worldwide, metastatic breast carcinoma being the second most lethal cancer among women. So, improvement of chemotherapeutic approaches to metastatic breast carcinoma is a goal of the utmost importance in the public health area. Since Rosenberg´s serendipitous discovery of cisplatin, a widely used antineoplastic agent, other metal-based antitumour compounds have been pursued with a view to overcome cisplatin´s severe nephrotoxicity and acquired resistance. One of the research lines along this subject concerns polynuclear chelates comprising cisplatin-like moieties linked by biogenic polyamine chains. The present study focuses on two dinuclear Pt(II) and Pd(II) chelates with spermine (Pt2Spm and Pd2Spm), probing their cellular impact (alone or in combination). The first aim of this work was to evaluate the antineoplastic properties of these complexes towards triple negative human breast carcinoma cells (MDA-MB-231), in sole administration and combined with the anti-mitotic agent Docetaxel (Taxotere®), regarding anti-proliferative, anti-invasive and anti-angiogenic capacities, in search for a synergistic interaction. Improved therapeutic schemes were sought, capable of increasing the survival rates for this poor prognosis cancer, hopefully circumventing chemoresistance to cisplatin-like agents. Promising anti-proliferative, anti-invasive and anti-angiogenic abilities were found for Pd2Spm/Docetaxel combinations. In addition, studies of drug–cell interactions in cancer model systems were pursued, since they are essential in the pre-clinical stage of rational drug design, which relies on a thorough understanding of the mechanisms underlying cytotoxic activity and biological effects, at a molecular level. With the use of complementary vibrational spectroscopy methods, the cellular impact of Pt2Spm and Pd2Spm was assessed, using cisplatin as a reference compound. The effects on cellular metabolism were monitored in MDA-MB-231 cells, by Raman and synchrotron-radiation infrared microspectroscopies, for different drug concentrations (2 – 8 µM) at a 48 h drug exposure. Multivariate data analysis was applied, unveiling drug- and concentration-dependent effects: apart from discrimination between control and drug-treated cells, a clear separation was obtained for the different agents studied – Pt(II) vs Pd(II), and mononuclear (cisplatin) vs polynuclear (Pt2Spm and Pd2Spm). Spectral biomarkers of drug action were identified, as well as the cellular response to the chemotherapeutic insult. The main effect of the tested compounds was found to be on DNA, lipids and proteins, the Pd(II) agent having a more significant impact on proteins while its Pt(II) homologue affected the cellular lipid content at lower concentrations. These results suggest the occurrence of distinct and unconventional pathways of cytotoxicity for these dinuclear polyamine complexes. Raman and FTIR microspectroscopies were confirmed as powerful non-invasive techniques to obtain unique spectral signatures of the biochemical impact and physiological reaction of cells to anticancer agents. Finally, the first neutron scattering study on human cells is reported, for addressing the subject of solvent slaving to a drug by probing intracellular water, with a view to ascertain variations upon drug exposure. Inelastic and quasi-elastic neutron scattering spectroscopy experiments with isotope labelling were performed, for monitoring this interfacial water response to cisplatin treatment (at 8 and 20 µM) in the same cell line (MDA-MB-231). Optical vibrational data were also obtained, for lyophilised cells. The intracellular water behaviour in the presence of cisplatin was found to be different from bulk water as well as from cytoplasmic water in drug-free cells. Concentration-dependent dynamical changes evidencing a progressive mobility reduction were unveiled between untreated and cisplatin-exposed samples, concurrent with variations in the native organisation of water molecules within the intracellular medium as a consequence of drug action. The results thus obtained yielded a clear picture of the intracellular water response to cisplatin and constitute the first reported experimental proof of a drug impact on the cytomatrix by neutron techniques. This is an innovative way of tackling a drug´s pharmacodynamics, searching for alternative targets of drug action (secondary drug targets).<br>Apesar de todos os esforços, o cancro continua a ser um problema de saúde mundial sendo o cancro de mama metastático o segundo mais letal em mulheres. Assim, melhorar as abordagens quimioterapêuticas no caso do cancro de mama metastático é de extrema importância para a saúde pública. Desde a descoberta da cisplatina por Rosenberg que esta passou a ser amplamente utilizada em oncologia. No entanto, devido à sua elevada nefrotoxicidade e resistência adquirida a procura de outros compostos inorgânicos com vista a ultrapassar estes problemas é de extrema importância. Uma das linhas de investigação na área é a inclusão de unidades semelhantes à cisplatina em quelatos polinucleares utilizando poliaminas biogénicas como ligandos. Este trabalho visa explorar o impacto celular de dois quelatos dinucleares de Pt(II) e de Pd(II) com espermina (Pt2Spm e Pd2Spm), tanto em administração isolada como em combinação com outros fármacos. O primeiro objetivo deste estudo foi avaliar as propriedades antineoplásicas destes complexos quando administrados isoladamente ou em combinação com o Docetaxel (Taxotere®), um agente anti-mitótico, numa linha celular humana de cancro de mama triplo negativo (MDA-MB231), avaliando a sua capacidade anti-proliferativa, anti-invasiva e anti-angiogénica, com vista à obtenção de sinergismo. O desenvolvimento de melhores esquemas terapêuticos poderá levar a um aumento da percentagem de sobrevivência para este tipo de cancro (de fraco prognóstico), contornando a resistência à quimioterapia para complexos semelhantes à cisplatina. Para a combinação Pd2Spm/Docetaxel foram obtidos efeitos anti-proliferativo, anti-invasivo e anti-angiogénico muito promissores. Foram ainda realizados estudos de interação fármaco-célula em modelos de cancro humano, sendo estes essenciais ao desenvolvimento racional de fármacos numa fase pré-clínica. A utilização de métodos complementares de espectroscopia vibracional permitiu determinar o impacto celular dos complexos Pt2Spm e Pd2Spm, usando a cisplatina como composto referência. O impacto sobre o metabolismo celular foi monitorizado para a linha MDA-MB231, por microespectroscopias de Raman e Infravermelho (com radiação de sincrotrão) utilizando diferentes concentrações de composto (2 – 8 µM) para um tempo de exposição de 48 h. O uso de análise multivariada dos dados obtidos permitiu identificar um nítido efeito dependente da concentração, tal como discriminar entre células controlo e células tratadas, assim como o impacto dos diferentes agentes testados – Pt(II) vs Pd(II) e mononuclear (cisplatina) vs polinuclear (Pt2Spm and Pd2Spm). Foram identificados biomarcadores espectrais da ação destes compostos, assim como a resposta celular devida à perturbação quimioterapêutica. O efeito principal dos complexos estudados verificou-se ao nível do DNA, dos lípidos e das proteínas, tendo o agente de Pd(II) um impacto mais significativo nas proteínas enquanto que o seu homólogo de Pt(II) afetou mais os lípidos (mesmo a baixas concentrações). Os resultados obtidos sugerem a ocorrência de vias distintas e não convencionais de citotoxicidade para estes complexos dinucleares com poliaminas. As microespectroscopias de Raman e FTIR foram confirmadas como sendo técnicas potentes e não-invasivas para obtenção de assinaturas espectrais do impacto bioquímico e reação fisiológica das células a agentes anticancerígenos. Finalmente, é reportado o primeiro estudo por difração de neutrões em células humanas, que visa abordar o condicionamento do solvente a um fármaco através da análise da água intracelular com o intuito de verificar as variações provocadas pela exposição ao agente quimioterapêutico. As experiências de espectroscopia de difração de neutrões, inelástica e quase-elástica, com marcação isotópica foram realizadas de modo a monotorizar a resposta da água interfacial ao tratamento com cisplatina (8 e 20 µM) na mesma linha celular (MDA-MB231) previamente investigada. Foram também obtidos dados de espectroscopia vibracional ótica para as células liofilizadas. Verificou-se que o comportamento da água intracelular na presença da cisplatina foi diferente do da água livre de constrangimentos, assim como da água citoplasmática em células não tratadas. As alterações dinâmicas dependentes da concentração evidenciaram uma redução de mobilidade progressiva (e dependente da concentração) entre as amostras não tratadas e as expostas à cisplatina, observadas em simultâneo às variações na estrutura nativa das moléculas de água no meio intracelular, como consequência da ação do fármaco. Os resultados obtidos permitiram obter uma imagem clara da resposta à cisplatina por parte da água intracelular, sendo esta a primeira prova experimental, mediante o uso de técnicas espectroscópicas com neutrões, do impacto de um fármaco na matriz celular. Este é um modo inovador de abordar a farmacodinâmica de um agente quimioterapêutico, na busca de recetores alternativos (alvos terapêuticos secundários).

碩士<br>國立臺北科技大學<br>有機高分子研究所<br>100<br>Breast cancer is responsible for the major cause of death in women. According to WHO’s report, the rate of occurance of breast cancer in Taiwanese women ranked second among the Asian countries. We established a novel screening platform for chemicals (pure compounds and extracts) which could inhibit the cell proliferation in cancer cells. In this platform, we used NIH/3T3 as control cell line. And, NIH/3T3-35 was generated by stably transfecting a novel gene and used as our cancer cell line. The MTT assay was performed to find an effective compound among the 1,600 chemical compounds screened for inhibition of mouse cancer cells and human cancer cells. The chemical compound #1736 was one of the selected compounds from the screening platform, which showed effective inhibition on human breast cancer cells. The objective of this study was to explore the mechanism of action for 1736, and its ability to induce apoptosis in MCF-7, a human breast cancer cell line. First, the MCF-7 cells were treated with different concentrations of 1736. The viability of cells depended on the dosage of 1736. Then, the cDNAs from the control and treated cells were hybridized with oligo-DNA microarray. The gene expression data from microarray were functionally annotated by using the bioinformatics tools, Database for Annotation, Visualization and Integrated Discovery (DAVID) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The results indicated that the MCF-7 treated with 1736 showed the functional gene pathways of apoptosis in human breast cancer cells, via JAK-STAT and p53 signaling pathways. The expression of DDIT3 gene was examined by reverse transcriptase polymerase chain reaction (RT-PCR) and the results were consistent with the cDNA microarray data. The expression of DDIT3 was found to be altered by 1736. DDIT3 gene regulates the cell differentiation; induce apoptosis when cells were subjected to ER stress. In addition to gene expression, western blot and immunocytochemistry analysis were performed to confirm the activation of DDIT3, at protein level. The results showed MCF-7 treated with 1736 increased the activation of DDIT3. In summary, 1736 can be further studied for developing a potential new anti-breast cancer drug.

Vitamin E is a group of naturally occurring fat soluble compounds which consists of eight distinct forms of tocopherols and tocotrienols. Although a well-defined physiological function of vitamin E is as an antioxidant, beneficial effects of individual vitamin E compounds on chronic human diseases such as cancer need to be better understood. Studies in this dissertation investigated potential application of gamma-tocopherol (gamma-T), gamma-tocotrienol (gamma-T3) or synthetic derivatives of tocotrienols as anticancer agents in comparison to alpha-tocopherol (alpha-T), its redox-silent acetic acid derivative (alpha-TEA) or alpha-tocotrienol (alpha-T3). Redox-silent derivatives of alpha- and gamma-T3; namely alpha-T3EA and gamma-T3EA exhibited potent anti-proliferative and proapoptotic activities in a murine mammary cancer cell line as well as in human breast cancer cell lines. Moreover, studies using human vascular endothelial cells in cell culture showed that the tocotrienol derivatives exhibited strong antiangiogenic activities which were markedly improved over those of the parent compounds. An antitumor efficacy study using the 66cl-4-GFP syngeneic mouse mammary tumor model showed that each tocotrienol derivative, when delivered in the diet, significantly suppressed mammary tumor growth; however serum and tissue concentrations of these novel compounds were lower than those of alpha-TEA, suggesting that the next generation of vitamin E derivatives will need to be modified to improve bioavailability. On the other hand, some natural-source vitamin E forms, especially gamma-forms, display anticancer activities without any chemical modification in both in vitro cell culture studies and in vivo animal models. Dietary delivery of gamma-T3 suppressed tumor growth in a syngeneic implantation mouse mammary cancer model by inhibiting cell proliferation and inducing apoptosis. Cell culture studies using human breast cancer cells showed that gamma-T3 triggered apoptosis by inducing endoplasmic reticulum (ER)-stress mediated by acid sphingomyelinase (ASMase) action. Activation of stress-activated mitogen-activated protein kinases (MAPKs), JNK and p38, was associated with gamma-T3-induced ER stress followed by upregulation of extrinsic death receptor-5 (DR5) expression in a CHOP transcription factor dependent manner. Gamma-T also triggered extrinsic apoptosis signaling by increasing DR5 mRNA, protein and cell surface expression levels followed by mitochondria-dependent apoptotic signaling. In agreement with in vitro studies, gamma-T delivered in the diet suppressed the tumor growth of MDA-MB-231-GFP human breast cancer cells in a xenograft model but the antitumor activity of gamma-T was hampered by co-administration of alpha-T. The preferential tissue retention of alpha-T over gamma-T could be overcome by use of sesamin, a dietary source of human cytochrome P450 inhibitor. Based on data presented, gamma-T and gamma-T3 show preclinical potential for cancer treatment either as single agents or in combination with other agents.<br>text

Tese de doutoramento em Bioquímica, na especialidade de Biologia Celular e Molecular, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra<br>O cancro continua a ser uma das principais causas de morte em todo o mundo. O cancro de mama, em particular, é um dos que apresenta a maior taxa de incidência em mulheres. Desta forma, novas abordagens para o tratamento do cancro de mama baseadas no desenvolvimento de compostos anticancerígenos promissores que permitam uma eficiente melhoria na sobrevivência do paciente, são de extrema importância. No presente trabalho, várias poliaminas biogénicas modificadas e correspondentes complexos de platina (Pt(II)) e paládio (Pd(II)) foram estudados relativamente ao seu comportamento conformacional, através de espectroscopia vibracional (infravermelho e Raman). Uma vez que os complexos de Pt(II) e Pd(II) têm demonstrado uma boa actividade anticancerígena, um conhecimento rigoroso do perfil estrutural destes compostos é essencial para compreendermos a base molecular da sua atividade citotóxica. A geometria all-trans foi reconhecida como sendo a mais favorável para todas as poliaminas alquiladas, em condições totalmente protonadas. Po outro lado, os respetivos complexos polinucleares apresentaram uma geometria estável muito semelhante àquela previamente obtida para os quelatos análogos com espermidina e espermina, contendo duas ou três unidades análogas da cisplatina (MCl2(NH3)2). A atividade antineoplásica destes compostos in vitro foi avaliada em linhas celulares humanas de cancro de mama (JIMT-1, L56Br-C1, MCF-7 e MDA-MB-231), assim como numa linha imortalizada não-neoplásica (MCF-10A), como sistema modelo. As células MCF- 10A mostraram ser menos afetadas pelos compostos testados, no entanto uma inibição significativa do crescimento e uma indução de morte celular (por apoptose) foram detetadas em algumas das linhas cancerígenas estudadas. As células L56Br-C1 foram as mais sensíveis a este tipo de tratamento. A norespermidina (NSpd) e o seu complexo de Pd(II) apresentaram um efeito antiproliferativo mais forte relativamente ao análogo de Pt(II). Verificou-se ainda que estes compostos reduziam a atividade celular da enzima ornitina descarboxilase na maioria das linhas celulares analisadas, para doses inferiores relativamente às do complexo de Pt(II). Além disso, o tratamento com NSpd ou com Pd-NSpd diminuíram a formação de colónias, i.e. a malignidade das células de cancro de mama, com maior eficiência do que o homólogo de Pt(II). Nenhum destes compostos aparentou atuar como uma genotoxina (na concentração de 25 μM). Relativamente aos análogos da espermina, a paladinação do N1,N11- bis(etil)norespermina (BENSpm) levou a um aumento da citotoxicidade, em contraste com a platinação do N1-metilciclopropil-N11-etilnorespermina (CPENSpm) que reduziu a citotoxicidade, o que pode ser explicado por diferenças na entrada celular dos dois quelatos. O BENSpm e o Pd-BENSpm apresentaram um efeito redutor face à população de células estaminais cancerígenas CD44+CD24- (avaliada por citometria de fluxo). O Pd-BENSpm foi o composto mais eficiente de todos os investigados relativamente à indução de danos no ADN e redução na formação de colónias. O Pt-CPENSpm e o Pd-Spm, por sua vez, mostraram ser os compostos menos tóxicos de todos os testados. O Pd-Spm diminuiu eficientemente os níveis intracelulares de glutationa, o que provavelmente foi uma consequência da sua inativação metabólica por conjugação a este tiol endógeno. Os resultados obtidos mostram que os complexos polinucleares de poliaminas com Pd(II) possuem uma atividade anticancerígena mais elevada do que os seus homólogos de Pt(II), podendo assim serem considerados promissores agentes terapêuticos inorgânicos contra o cancro de mama, acoplando uma maior eficiência a uma toxicidade mais reduzida.<br>Worldwide, cancer is still one of the major leading causes of death. In particular, breast cancer is the one presenting the highest incidence rates in women. Therefore, new approaches for breast cancer treatment based on the development of promising anticancer agents that can efficiently improve patient survival are of utmost relevance. In the present work, several modified biogenic polyamines and their newly synthesised Pt(II) and Pd(II) complexes were studied regarding their conformational behaviour by vibrational spectroscopy (infrared and Raman). Since Pt(II) and Pd(II) complexes have shown good anticancer activity, an accurate knowledge of the structural profile of these compounds is essential for understanding the molecular basis of their cytotoxic activity. The all-trans geometry was found to be favoured for all the alkylated polyamines, in their totally protonated state. In the other hand, their polynuclear complexes presented a stable geometry very similar to that previously obtained for the analogous chelates with spermidine and spermine, comprising two or three cisplatin-like (MCl2(NH3)2) moieties. The in vitro cytotoxic activity of these compounds was then evaluated in established human breast cancer cell lines (JIMT-1, L56Br-C1, MCF-7 and MDA-MB-231) and in one normal-like immortalised breast cell line (MCF-10A) as model systems. The MCF-10A cells were found to be least affected by the tested compounds, while significant growth inhibition and apoptotic cell death was observed for some of the cancer cell lines, L56Br-C1 being the most sensitive to this type of treatment. Norspermidine (NSpd) and its Pd(II) complex were generally shown to have a stronger antiproliferative effect, as compared to its Pt(II) analogue. Also they were found to reduce the cellular activity of the growth-related enzyme ornithine decarboxylase to a lower level than the Pt(II) complex, in most of the cell lines examined. Moreover, treatment with NSpd or Pd- NSpd decreased colony formation, i.e. the malignancy of the breast cancer cells, to a much larger extent than the Pt(II) counterpart. None of these agents appeared to act as a genotoxin at a 25 μM concentration. Regarding the spermine analogues, palladination of N1,N11-bis(ethyl)norspermine (BENSpm) led to enhanced cytotoxicity, in contrast to platination of N1-cyclo-propylmethyl- N11-ethylnorspermine (CPENSpm), that reduced cytotoxicity, which may be explained by differences in the cellular uptake of the chelates. BENSpm and Pd-BENSpm treatment reduced the putative cancer stem cell population CD44+CD24- (evaluated by flow cytometry). Furthermore, Pd-BENSpm was the most efficient compound of those investigated regarding induction of DNA damage and decrease in colony formation. Pt-CPENSpm and Pd-Spm, in the other hand, were shown to be the least toxic compounds of all tested. Pd-Spm efficiently reduced the cellular glutathione levels, which probably was a consequence of its metabolic inactivation by conjugation to this endogenous thiol. Overall, the results show that the polynuclear Pd(II)-polyamine complexes display a stronger anticancer effect than their Pt(II) homologues, which renders them promising new metal-based therapeutic agents against breast cancer, coupling increased efficiency to a slightly lower toxicity.<br>FCT - SFRH/BD/46364/2008

Cancer is the second leading cause of death worldwide being responsible for 9.6 million deaths of a total of 18.1 million new cases in 2018. In particular, breast cancer is the most commonly diagnosed and the leading cause of death in women. Among the several types of breast cancers, the triple negative breast cancer, which is estrogen receptor negative (ER-), progesterone receptor negative (PR-) and human epidermal growth factor receptor 2 negative (HER2-) is known for its typical highly metastatic profile, with inferior prognosis and for which there is still no available effective targeted therapy. Thus, the investment in the research of new chemotherapeutic agents in this area is crucial and imperative. Within this PhD project, six new organic ligands and eighteen new ruthenium(II) cyclopentadienyl compounds were synthesized and fully characterized. These complexes were divided in three different families with the general formula [Ru(Cp)(P)(bipy)]+, where cyclopentadienyl (Cp), phosphane (P) and bipyridyl (bipy) moieties were functionalized with different groups in order to include a macromolecular ligand (polylactide - PLA and/or polyethylene glycol - PEG) and a biomolecule (biotin or curcumin) to enhance the selectivity of compounds to cancer cells, exploring both passive and active cancer targeting strategies. To assess the biological potential of the new compounds an array of in vitro studies aiming at establishing structure-activity relations was performed. The studies in hormone and nonhormone-responsive cancer cell lines encompassed the assessment of cytotoxicity, cell death, intracellular distribution (drug internalization) and anti-metastatic ability (colony formation assay) of compounds. Multidrug resistance for selected compounds on the main ABC transporters was also studied by flow cytometry. Finally, compounds’ toxicity was assessed using a zebrafish model and the lead compounds were subjected to preliminary in vivo studies using a nude mice animal model bearing triple negative breast cancer orthotopic tumors where their toxicity and efficacy on primary tumors and metastases was evaluated.<br>Fulbright com o apoio da FCT