Conselho Nacional de Desenvolvimento Científico e Tecnológico / Os mecanismos de ação citotóxica do inibidor de histona deacetilase LBH589 foram investigados em associação com o quimioterápico cisplatina (CDDP) em duas linhagens derivadas de câncer de pulmão de não pequenas células (CPNPC). Os resultados foram analisados em relação ao tipo de morte celular associada às alterações em enzimas relacionadas ao metabolismo energético e a via glicolítica. Para realização do trabalho, foram utilizadas as linhagens tumorais A549 (selvagem para o gene de p53) e Calu-1 (nulo para o gene de p53) tratadas com LBH589 em combinação ou não com CDDP. Foram realizadas curvas de tempo e dose-resposta com as drogas isoladamente pelo ensaio de viabilidade celular (MTT) nas duas linhagens para a escolha das melhores condições para o nosso estudo. As condições dos tratamentos isolados com redução da viabilida celular menores que o IC50 de cada fármaco foram selecionados para realização dos tratamentos combinados. As avaliações de apoptose foram realizadas por citometria de fluxo pelo ensaio de Anexina V/PI, e com a marcação de proteínas por Western Blotting. As proteínas relacionadas a via glicolítica foram avaliadas por Western Blotting e a expressão de RNAm por qPCR. Os resultados demonstraram que o LBH589 combinado a CDDP foi capaz de induzir apoptose em 70% das células (Calu-1) e 54,9% (A549) no tempo de 24 horas, e 90% (calu-1) e 62,1% (A549) em 48 horas, independendo, portanto, do status da p53. Os níveis de expressão de enzimas relacionadas com o metabolismos energético também sofreram alterações nos tratamentos estudados. O LBH589 induziu aumento de cerca de 4x dos níveis de RNAm de HK isoformas I e II em ambas as linhagens. Houve também um aumento na expressão proteica das isoformas de HK I e II. Outras enzimas relacionadas a via glicolítica como PFKP, PKM2 e LDHA foram analisadas e apresentaram redução da expressão proteica, principalmente na presença do LBH589. A combinação da CDDP com LBH589 parece ser promissora para o tratamento de CPNPC induzindo apoptose através de alterações no metabolismo energético tumoral. / The cytotoxic mechanisms of action of the histone deacetylase inhibitor LBH589 was investigated in association with cisplatin in two cell lines derived from non-small lung cancer (NSCLC). The results were analyzed based on the type of the cell death associated with alterations in enzymes related to energy metabolism and glycolytic pathway. To perform the study, we used two non-small lung tumor cell lines, A549 (wild type for the p53 gene) and Calu-1 (null for p53 gene) treated with LBH589 in combination with cisplatin or not. Assays for time and dose responses were performed through the cell viability analysis (MTT) for each drug separately in both cell lines to choose the best conditions for our study. The conditions of isolated treatments with reduced viability lower than the IC50 of each drug were selected for carrying out the combined treatments. Assays for apoptosis detection by flow cytometry were performed by Annexin V / PI, the expression of proteins related to apoptosis by Western Blotting. The expression of glycolytic related proteins were performed by Western blotting and their mRNA expressions by qPCR. The results demonstrated that the combined LBH589 plus CDDP was able to induce apoptosis in 70% of cells -Calu-1- and 54.9% -A549- in 24 hours, and 90% (Calu-1) and 62.1 % (A549) in 48 hours regardless of the p53 status. The expression levels of enzymes related to energy metabolism also presented changes in the studied treatments. The LBH589 increase of about 4x the HK isoforms I and II mRNA levels in both cell lines. There was also an increase in the expression isoforms I and II of HK protein. Other enzymes related to the glycolytic pathway as PFKP, PKM2 and LDHA were analyzed and we observed reduced protein expression, especially in the presence of LBH589. The combination of CDDP with LBH589 appears promising for the treatment of NSCLC inducing apoptosis via alterations in energy metabolism tumor.

<p>The serendipitous discovery of the potent cytotoxic properties of cisplatin brought about a revolution in the treatment of certain types of cancer, but almost fifty years later, there still remain unknown areas in the chemistry of cisplatin. There are questions regarding which form of the drug reaches its DNA target, or why certain DNA sequences are more preferred than others for reaction with cisplatin. The work presented here aims to address some of these problems, using quantum chemical calculations to complement and interpret available experimental data.</p><p>Cisplatin's activation reactions are explored by Density Functional Theory (DFT) on two model systems, one solely using a self-consistent reaction field (SCRF) for modeling bulk water, and one including an additional partial solvation shell of water molecules. It is concluded that adding explicit solvation provides a better picture than using SCRF solvation alone. The energy surface supports the view that the active form of cisplatin is the monoaquated form.</p><p>The activation reactions of the cisplatin-derived drug, JM118, are investigated using DFT and SCRF calculations using three solvation model systems. The results show a slower rate of hydrolysis for the first reaction, and a faster rate for the second, suggesting diaquated JM118 as the main DNA binding form of the drug.</p><p>Diaquated cisplatin's first and second reaction with guanine and adenine are studied using DFT and SCRF solvation. Cisplatin's propensity toward guanine in the first substitution is explained by larger stabilisation energy for the initially formed complex and by favoured kinetics. For the second substitution, higher stability in complexation with guanine over adenine is ascribed as the main factor favouring guanine over adenine substitution. This provides the first explanation for the predominance of 1,2-d(GpG) over 1,2-d(ApG) adducts, and the direction specificity of the 1,2-d(ApG) adducts.</p>

Au cours de ces années de Doctorat, nous avons étudié les effets ototoxiques de certains médicaments et les moyens de prévenir les surdités neuro-sensorielles quils peuvent induire. Parmi ces molécules, nous nous sommes concentrés sur les plus couramment utilisées en pratique clinique : les antibiotiques de la famille des aminoglycosides et le cisplatine, un agent anti-cancéreux. Lintroduction de notre travail replace la surdité dans son contexte de santé publique. En particulier, nous décrivons pourquoi les médicaments ototoxiques sont utilisés et dans quelles circonstances. Nous présentons la structure de loreille interne et nous tentons dexpliquer sa vulnérabilité aux molécules ototoxiques. Nous abordons ensuite les moyens de prévention et/ou de traitement de ces atteintes neuro-sensorielles pharmaco-induites. Outre les moyens classiques de prévention, que sont les facteurs trophiques et les antioxydants, nous décrivons de nouvelles voies dapproche que sont les voies de signalisation impliquant la protéine kinase C ou la cascade dactivation RhoA/ROCK. La présentation de notre travail original sarticule autour de deux parties. Dans la première partie, nous rapportons les résultats obtenus au cours de notre étude de la toxicité des aminoglycosides et du cisplatine chez la souris et le cobaye in vivo. Nous avons mis en évidence une différence de vulnérabilité significative entre ces deux espèces face à lagression ototoxique. Cette différence existe au niveau fonctionnel, mis en évidence par létude des potentiels évoqués auditifs, et au niveau anatomique, étudié en histologie et en immunohistochimie. Nous en discutons les implications en recherche et en pratique clinique. Dans la seconde partie, nous étudions les moyens de prévenir cette surdité in vivo et in vitro. Nous avons utilisé un modèle de surdité par aminoglycoside chez le cobaye. Nous avons testé et validé une technique de perfusion intra-cochléaire in vivo. Nous avons observé les effets de deux molécules expérimentales : la Bryostatine 1, un activateur de la protéine kinase C, et un inhibiteur de la voir RhoA-ROCK. Leffet protecteur de ces molécules est actuellement limité au ganglion spiral, dont la survie est essentielle à tout traitement dimplantation prothétique et de réadaptation. Nous discutons des perspectives en médecine humaine dans notre conclusion. In this work, we focused our attention on the effects of main ototoxic drugs i.e. aminoglycosides and cisplatin in mammals. We identified new avenues for the prevention of this toxicity. In the introduction, we described how and why ototoxic drugs are used. We then described potential otoprotective strategies in neurosensory deafness. Among them, trophic factors and antioxidant molecules have been widely used. New otoprotective approaches do exist, implying the protein kinase C or RhoA/ROCK signalling. Our original work was presented in two parts. In the first part, we reported the in vivo effects of aminoglycosides and cisplatin in two mammalian species: mice and guinea pigs. Contrarily to guinea pigs, evidence of mice resistance to ototoxicity was found at a functional level, assessed by auditory brainstem responses, and at an anatomical level, studied by immunohistochemistry. We discussed the implication of such differences in research and in clinical practice. In the second part, we studied the effect of two potential otoprotective molecules: Bryostatine 1, an activator of the protein kinase C, and Y-27632, a Rho kinase inhibitor. We showed that these molecules are protecting spiral ganglion neurons both in vitro and in vivo. Survival of spiral ganglion neurons is crucial in the management and rehabilitation of deafness. The potential perspectives of these results in human medicine were discussed.

The serendipitous discovery of the potent cytotoxic properties of cisplatin brought about a revolution in the treatment of certain types of cancer, but almost fifty years later, there still remain unknown areas in the chemistry of cisplatin. There are questions regarding which form of the drug reaches its DNA target, or why certain DNA sequences are more preferred than others for reaction with cisplatin. The work presented here aims to address some of these problems, using quantum chemical calculations to complement and interpret available experimental data. Cisplatin's activation reactions are explored by Density Functional Theory (DFT) on two model systems, one solely using a self-consistent reaction field (SCRF) for modeling bulk water, and one including an additional partial solvation shell of water molecules. It is concluded that adding explicit solvation provides a better picture than using SCRF solvation alone. The energy surface supports the view that the active form of cisplatin is the monoaquated form. The activation reactions of the cisplatin-derived drug, JM118, are investigated using DFT and SCRF calculations using three solvation model systems. The results show a slower rate of hydrolysis for the first reaction, and a faster rate for the second, suggesting diaquated JM118 as the main DNA binding form of the drug. Diaquated cisplatin's first and second reaction with guanine and adenine are studied using DFT and SCRF solvation. Cisplatin's propensity toward guanine in the first substitution is explained by larger stabilisation energy for the initially formed complex and by favoured kinetics. For the second substitution, higher stability in complexation with guanine over adenine is ascribed as the main factor favouring guanine over adenine substitution. This provides the first explanation for the predominance of 1,2-d(GpG) over 1,2-d(ApG) adducts, and the direction specificity of the 1,2-d(ApG) adducts.

Even though anti‐cancer chemotherapy has been continuously improved during the last decades. problems with adverse effects and drug resistance still constitutes a considerable obstacle and sets a demand for new effective treatment options. Tissue homeostasis in multi‐cellular organisms is maintained through intrinsic cell death, apoptosis, which removes unwanted or damaged cells. Disrupted apoptosis is an important factor in tumorgenesis and drug resistance, therefore induction or restoration of apoptotic pathways is also important for the treatment of cancer. Several naturally occurring bacterial toxins have the ability to induce apoptosis and could thus be candidates to complement or improve the therapeutic effect of other anticancer drugs. The bacterial toxins, adenylate cyclase (AC) toxin from Bordetella pertussis, α‐toxin from Staphylococcus aureus and verotoxin‐1 (VT‐1) from Escherichia coli were investigated for their ability to induce apoptosis in different tumor cell lines. Toxin induction of cell death was investigated by cell viability assays, end‐stage apoptosis induction by DNA‐fregmentation (TUNEL) assay. Toxin receptor expression and signal transduction pathways to apoptosis were investigated by flow cytometry, caspase enzyme activity assays and western blot. Immunohistochemistry was used for identification of toxin receptor expression in tumor tissue samples. AC‐toxin was cytotoxic and induced apoptosis in cultured malignant plural mesothelioma (MPM) and small‐cell lung cancer (SCLC) cells. Low‐toxic concentrations of AC‐toxin enhanced cisplatin cytotoxicity and apoptosis in both cell lines. MPM‐cells with acquired cisplatin resistance were more sensitive to α‐toxin than the less resistant parental MPM cell line. A low‐toxic concentration of α‐toxin re‐sensitized resistant MPM cells to cisplatin cytotoxicity by apoptosis induced through the mitochondrial pathway without detectable activation of common up‐stream apoptosis signalling proteins. VT‐1 was highly cytotoxic and induced apoptosis in globotriosylceramide (Gb3) ‐expressing glioma, breast cancer and non‐small‐cell lung cancer (NSCLC) cells but was not cytotoxic to non‐Gb3‐expressing cells. PPMP, an inhibitor of glucosylceramide synthesis which makes exposed cells unable to synthesize Gb3 rendered Gb3‐expressing cells resistant to VT‐1. MPM cells with acquired‐cisplatin resistance expressed Gb3 in contrast to the absent of expression in the less resistant parental cell line. Gb3, could however be up‐regulated by cisplatin in Gb3‐negative MPM‐cells. Presence of a low‐toxic concentration of VT‐1 potentiated cisplatin‐induced cytotoxicity and apoptosis in the cisplatin‐resistance MPM cell line. VT‐1 was a potent inducer of apoptosis, probably via stress‐induced Mitogen‐activated protein kinase (MAPK)‐signaling involving c‐Jun N‐terminal kinase (JNK) and p38, leading to disruption of the mitochondrial membrane integrety, activation of caspase‐9 and ‐3, and ultimately DNA fragmentation and cell death. Gb3 expression was demonstrated in clinical specimens of glioblastoma and breast cancer making these tumor types interesting for further VT‐1 studies. We conclude that bacterial toxins may be used to induce apoptosis in several types of cancer cells. Low concentrations of verotoxin‐1 and α‐toxin may potentially be used to overcome acquired cisplatin‐resistance in cancer patients.

The scientific progress about the important chemotherapeutic drug substance cisplatin (CDDP) and its function has often been rendered by data difficult to interpret, and still many questions about its mode of action remains to be clarified by the scientific community. However, studies of CDDP possess a high complexity due to; i) low intracellular concentration, ii) many potential biomolecule targets, iii) poor or unknown stability of the intact drug and its biomolecule adducts and iv) complex and varying sample matrices. Metallomic studies, using advanced analytical techniques may contribute to clarify the interactions between CDDP and intracellular biomolecules. For a successful outcome sample preparation conditions as well as separation and detection techniques must be carefully selected and optimized to achieve accurate results and correct interpretation of data.         This thesis describes some new and improved analytical methodologies for characterizing the Pt metallome in CDDP-exposed malignant cells. The developed methods are based on powerful liquid chromatography (LC) methods hyphenated to sensitive detection by inductively coupled plasma- (ICP) and electrospray ionization mass spectrometry (ESIMS). Consideration has also been taken about sample preparation conditions.         By selecting “chemically inert” sample preparation (cell lysis by osmosis) and separation (using only nonreactive or no additatives) conditions we could avoid the formation of platinum artifact compounds previously described in the literature (Paper I and II). Using oxygen containing organic solvents with high boiling points (dimethylformamide; DMF, 1,4-dioxane, n-propanol and ethanol) as alternatives to acetonitrile in the LC separations, significant improvements were achieved in ICPMS sensitivity and robustness. When evaluated in combination with chromatographic performance and ESIMS detection the overall best performance was achieved with n-propanol (Paper II, III and IV). From the studies in Paper II we could show that free intact CDDP can be found in malignant cells, as supporting evidence for passive or endocytotic uptake of the drug and further estimate a half-life for intracellular CDDP to about 15 minutes. Such data has not been shown before. In Paper V, the above improved LC methods were used to demonstrate differences in the platinum and cupper metallome from sensitive and resistant T289 melanoma cells exposed to CDDP at near clinical levels.         In a wider perspective we have shown the potential of using hydrophilic liquid interaction chromatography (HILIC) hyphenated to ICPMS detection as a general approach for analysis of hydrophilic metallo-compounds (Paper II). Taking advantage of the superior ICPMS performance using n-propanol gradients for reversed phase liquid chromatography (RPLC) possess a true alternative and /or complimentary technique to size exclusion chromatography (SEC) commonly applied within metallomic studies of biomolecules (Paper V). Using n-propanol in HILIC as well as in RPLC enables parallel detection by ICP- and ESIMS using only one set of chromatographic parameters (Paper III and IV), something commonly called for by scientists in the field.

El cisplatí, PtCl2(NH3)2, ha estat una de les drogues més utilitzades en la quimioteràpia del càncer des del descobriment de la seva activitat. Però degut a la seva alta toxicitat i greus efectes secundaris, s'han sintetitzat nous compostos amb la finalitat de reduir aquests inconvenients.En aquest sentit, el treball desenvolupat en aquesta tesi doctoral ha estat la síntesi i caracterització de tretze complexos de Pt(II) amb la finalitat d'estudiar llur activitat antitumoral. Aquests complexos presenten unes característiques estructurals comunes: geometria cis, dos lligands làbils de tipus clorur i un lligand diaminoquelatant derivat dels àcids d,l-2,3-diaminopropiònic (Hdap) i d,l-2,4-diaminobutíric (Hdab). S'han dissenyat unes estratègies sintètiques a partir de les quals els lligands han estat funcionalitzats amb diferents grups de tipus éster, aminoàcid i peptídic: Etdap·2HCl, Etdab·2HCl, [(dap-Metala)·2CF3COOH], [(dab-Metala)·2CF3COOH], [(dap-phe)·2CF3COOH], [(dab-phe)·2CF3COOH], [(dap-Mettrp)·2CF3COOH], [(dab-Mettrp)·2CF3COOH], [(dap-ASTTTNYT-NH2)·2CF3COOH], essent Metala= éster metílic de L-alanina, phe= L-fenilalanina, Mettrp= éster metílic del L-triptofà. Aquests lligands diaminoquelatants s'han utilitzat per sintetitzar els corresponents complexos de Pt(II): PtCl2(Hdap), PtCl2(Hdab), PtCl2(Etdap), PtCl2(Etdab), PtCl2(dap-Metala), PtCl2(dab-Metala), PtCl2(dap-ala), PtCl2(dab-ala), PtCl2(dap-phe), PtCl2(dab-phe), PtCl2(dap-Mettrp), PtCl2(dab-Mettrp), PtCl2(dap-ASTTTNYT-NH2).A través de diferents tècniques i assaigs biològics (dicroisme circular, electroforesi en gel d'agarosa, microscopia de forces atòmiques, citometria de flux, assaigs de proliferació cel·lular) s'ha pogut demostrar l'activitat antitumoral d'aquests compostos. A través de la tècnica de dicroisme circular (DC) s'ha pogut demostrar que els lligands lliures no interaccionen covalentment amb el DNA de Calf Thymus i no modifiquen l'estructura secundària de la doble hèlix. En canvi, els respectius complexos han demostrat tenir capacitat per interaccionar amb el DNA i modificar la seva estructura secundària. Els complexos PtCl2(Hdap), PtCl2(Hdab) i PtCl2(dab-phe) mostren un comportament similar al cisplatí, generant adductes cis-bifuncionals que distorcionen la doble hèlix de forma no desnaturalitzant amb obertura de la doble cadena. Els complexos PtCl2(Etdap), PtCl2(Etdab), PtCl2(dap-ala), PtCl2(dab-ala), PtCl2(dap-Metala), PtCl2(dab-Metala), PtCl2(dap-phe), PtCl2(dap-ASTTTNYT-NH2) quan interaccionen amb el DNA generen un canvi en la conformació del DNA de la forma B a la forma C, produint-se un augment de la curvatura de l'hèlix per rotació de les bases nitrogenades. En aquests estudis s'ha comprovat que l'estructura del complex influeix en l'efecte generat sobre l'estructura secundària de l'àcid nucleic. En primer lloc, existeix una diferència en el comportament en funció del tamany del lligand diaminoquelatant, de manera que els complexos amb el lligand (dab) provoquen un efecte més remarcable. També s'observa aquest canvi de comportament al passar dels complexos que tenen el grup funcional esterificat als que el tenen protonat. D'aquesta manera, s'observa un major efecte sobre l'estructura secundària del DNA en aquells complexos que tenen el lligand diaminoquelatant de tres metilens (dab) i amb el grup carboxilat terminal protonat.Per tal de modelitzar la interacció d'aquests complexos amb el DNA, s'ha estudiat la interacció d'aquests compostos de Pt(II) amb 5'-GMP a través de RMN-1H, observant la variació dels senyals corresponents al H8 de 5'-GMP. Així s'ha pogut demostrar que aquests compostos interaccionen amb la 5'-GMP a través d'un enllaç covalent Pt-N7, de la mateixa manera a com interacciona el cisplatí.A través d'electroforesi en gel d'agarosa i microscopia de forces atòmiques (AFM) s'ha pogut determinar l'efecte que generen els lligands lliures i els respectius complexos de Pt(II) sobre l'estructura terciària del plasmidi pBR322. Els lligands provoquen un augment de l'agregació de les molècules de DNA i un lleuger augment de la compactació de l'estructura terciària. Aquests resultats s'atribueixen a la capacitat d'aquests compostos a interaccionar per pont d'hidrogen amb el DNA. Els corresponents complexos de Pt(II) provoquen un augment de l'agregació i una important compactació, degut per una banda a la capacitat de l'àtom de Pt a interaccionar covalentment amb el DNA, i per altra banda, a la capacitat del lligand a interaccionar per pont d'hidrogen amb l'àcid nucleic.Finalment s'ha estudiat l'activitat citotòxica d'aquests complexos de Pt(II) en diferents línies cel·lulars: A431 (línia de carcinoma epidermoide), HeLa (línia de carcinoma de coll d'úter) i HL-60 (línia promielocítica de leucèmia). Els complexos moderadament solubles en aigua, PtCl2(Hdap), PtCl2(Hdab), PtCl2(dap-ala), PtCl2(dab-ala), PtCl2(dap-phe) i PtCl2(dab-phe), han demostrat ser actius. L'activitat depèn de la concentració de complex, del temps d'incubació i de la línia cel·lular. Per temps d'incubació alts i concentracions de complex elevades s'observa la màxima activitat. Els complexos de l'alanina, PtCl2(dap-ala) i PtCl2(dab-ala), són els que mostren més activitat, mentre que els compostos de la fenilalanina són els menys actius, degut probablement a la voluminositat del lligand, la qual pot impedir o dificultar el transport del compost a través de la membrana cel·lular.L'activitat citotòxica dels complexos insolubles en aigua, PtCl2(Etdap) i PtCl2(Etdab), queda bloquejada per l'elevada concentració de DMSO (12%) necessària per solubilitzar els compostos. Aquests resultats permeten deduir que la presència d'un 12% de DMSO anul·la l'activitat d'aquests complexos, ja que el DMSO pot coordinar-se amb el Pt ocupant les posicions làbils del complex i evitant que es pugui coordinar amb el DNA.Els assaigs de proliferació cel·lular del complex PtCl2(dap-ASTTTNYT-NH2) i del pèptid lliure ASTTTNYT-NH2 han demostrat que ambdós compostos són actius. Tot i això, l'activitat del complex és superior a la del pèptid lliure, ja que el Pt pot interaccionar covalentment amb el DNA i augmentar l'efecte citotòxic. Per tant, el complex presenta un lligand portador biològicament actiu que pot transportar el metall a través de la membrana cel·lular i facilitar així la seva interacció amb el DNA.A través de la tècnica de citometria de flux s'ha comprovat que en tots els casos la mort cel·lular produïda pels complexos ha estat per apoptosi.Per últim, s'ha sintetitzat i caracteritzat un complex trinuclear de Pt(II), {[Pt(Me2Bpy)2][PtCl2(Me2Bpy)]2}, essent Me2Bpy= 4,4'-dimetil-2,2'-dipiridil. La resolució de la seva estructura per difracció de Raig-X ha permès determinar l'existència d'una interacció intramolecular Pt-Pt de 3.474 Å. / Cisplatin, PtCl2(NH3)2, has been one of the most used drugs for the cancer chemotherapy. However, due to its high toxicity and important side effects, new complexes have been synthesized in order to reduce these disadvantages.In that way, the work presented in this thesis is the synthesis and characterization of 13 Pt(II) complexes in order to study their antitumor activity. These complexes present some common traits: cis geometry, two labile chloride ligands, and one diamine chelating ligand derived from d,l-2,3-diaminopropionic acid (Hdap) and d,l-2,4-diaminobutyric acid (Hdab). These ligands have been derived using new synthetic strategies with different functional groups as ester, aminoacid and peptide type: Etdap·2HCl, Etdab·2HCl, [(dap-Metala)·2CF3COOH], [(dab-Metala)·2CF3COOH], [(dap-phe)·2CF3COOH], [(dab-phe)·2CF3COOH], [(dap-Mettrp)·2CF3COOH], [(dab-Mettrp)·2CF3COOH], [(dap-ASTTTNYT-NH2)·2CF3COOH], being Metala= methyl ester of L-alanine, phe= L-phenylalanine and Mettrp= methyl ester of L-triptophan. These ligands have been used for preparing the following Pt(II) complexes: PtCl2(Hdap), PtCl2(Hdab), PtCl2(Etdap), PtCl2(Etdab), PtCl2(dap-Metala), PtCl2(dab-Metala), PtCl2(dap-ala), PtCl2(dab-ala), PtCl2(dap-phe), PtCl2(dab-phe), PtCl2(dap-Mettrp), PtCl2(dab-Mettrp), PtCl2(dap-ASTTTNYT-NH2).Using different techniques and biologic assays (circular dichroism, agarose gel electrophoresis, atomic force microscopy, flow cytometry, cell proliferation assays) the antitumor activity of these complexes has been demonstrated.With circular dichroism it has been demonstrated that free ligands do not covalently bind Calf Thymus-DNA and do not modify its secondary structure. In constrast, the corresponding Pt(II) complexes are able to interact with DNA and modify its secondary structure. Complexes PtCl2(Hdap), PtCl2(Hdab) and PtCl2(dab-phe) behave in the same direction as cisplatin, forming cis-bifunctional intrastrand adducts that probably open the double helix. The other complexes, PtCl2(Etdap), PtCl2(Etdab), PtCl2(dap-ala), PtCl2(dab-ala), PtCl2(dap-Metala), PtCl2(dab-Metala), PtCl2(dap-phe), PtCl2(dap-ASTTTNYT-NH2), interact with DNA generating a B&#61614;C transformation with increasing winding of the double helix by rotation of the bases. The influence of the complex structure in the effect on the secondary structure of DNA has been proved. Firstly, a difference in the behaviour exists depending on the ring size, so complexes with (dab) ligand cause bigger modifications. This kind of behaviour is also seen between complexes with ester groups at the C-terminus and complexes with carboxylic group. Thus, there is a greater effect in the secondary structure of the DNA in those complexes that have the (dab) ligand and a protonated carboxylate group.The interaction between the platinum complexes and 5'-GMP has been studied by 1H-NMR, following the variation of H8 signals from 5'-GMP. It has been demonstrated that all complexes covalently interact with 5'-GMP through the N7 of the base, as cisplatin do.The effect generated by free ligands and their Pt(II) complexes in the tertiary structure of pBR322 DNA has been determined by agarose gel electrophoresis and atomic force microscopy (AFM). Generally, the free ligands studied generate an increase of DNA aggregation and a slight coiling of the double helix, due to their possibility of forming hydrogen bonds with the DNA bases through different functional groups of their structure. The Pt(II) complexes generate an important coiling and aggregation of DNA, probably due to the covalent binding Pt-DNA and hydrogen bond interactions through their ligands.The cytotoxic activity of the Pt(II) complexes has been evaluated through cell proliferation assays in A431, HeLa and HL-60 cell lines using different incubation times (24h, 48h, 72h). The water-soluble complexes, PtCl2(Hdap), PtCl2(Hdab), PtCl2(dap-ala), PtCl2(dab-ala), PtCl2(dap-phe) i PtCl2(dab-phe), show antitumor activity in a dose- and time- dependent manner, gradually decreasing the cell survival percentage when the complex concentration increase, in the same direction as carboplatin. All these complexes present the highest activity at long incubation times and high complex concentration. The alanine complexes, PtCl2(dap-ala) and PtCl2(dab-ala) are the most active and those of phenylalanine are the less active, probably due to the ligand size, which can make its transport through cell membrane more difficult.The water-insoluble complexes, PtCl2(Etdap) and PtCl2(Etdab), apparently do not show antitumor activity due to the presence of 12% DMSO necessary por solving these complexes. In these conditions they do not cause a decrease in cell survival. The DMSO could interact with platinum, displacing the labile ligands of the complex and not allowing it to interact with DNA. Complex PtCl2(dap-ASTTTNYT-NH2) is also insoluble in water, but can be dissolved in 2% DMSO solution. Cell proliferation assays show that the free peptide, ASTTTNYT-NH2, and the platinum complex are both active. The activity of the complex is higher than the free peptide, probably because platinum can covalently interact with DNA and increase the cytotoxic effect. This result suggest that the ligand can act as a carrier ligand and make the interaction metal-DNA be easier.Flow cytometry assays have demonstrated that cell death generated by the complexes synthesized is apoptosis. Agarose gel electrophoresis of extracted DNA show a classical 200-base pair integer oligonucleosome ladder.A new trinuclear Pt(II) complex, {[Pt(Me2Bpy)2][PtCl2(Me2Bpy)]2} being Me2Bpy= 4,4'-dimethyl-2,2'-dipyridyl, has been synthesized and characterized. The X-ray diffraction of its structure shows the existence of an intramolecular Pt-Pt interaction of 3.474 Å.

The PAX gene family of transcription factors plays a prominent role during embryogenesis however can be aberrantly re-activated during tumorigenesis and contributes to the malignant phenotype. / During embryonic kidney development, PAX2 exerts an anti-apoptotic function however its expression typically attenuates during the post-natal period. On the other hand, PAX2 aberrant expression is observed in the majority of Renal Cell Carcinomas (RCC). RCC is resistant to chemotherapy; up-regulation of anti-apoptotic genes is recognized to contribute to tumor resistance to chemotherapy. We hypothesized that the anti-apoptotic effect of the PAX2 gene that is expressed in RCC cells contributes to RCC and their resistance to chemotherapy-induced cell death. / Human embryonic kidney (HEK293) cells transfected with a PAX2 expression vector and exposed to cisplatin, were protected from apoptosis compared to control cells. Conversely, murine collecting duct cells stably transfected with PAX2 antisense cDNA had twofold increases in cisplatin-induced apoptosis. Similarly, PAX2 knockdown using PAX2 siRNA in RCC cells CAKI-1 and ACHN enhances cisplatin-induced apoptosis in vitro. / To test the combination of PAX2 expression silencing and cisplatin treatment in vivo we developed a model of renal tumors by injecting ACHN cells as a xenograft under the skin of nude mice. I showed that a PAX2 shRNA successfully knocks down PAX2 mRNA and protein levels in a RCC cell line (ACHN). ACHN cells stably transfected with shRNAs targeted against the PAX2 homeodomain, are more susceptible to cisplatin-induced caspase-3 activation than the control ACHN cell line. Furthermore, growth of subcutaneous ACHN/shPAX2 xenografts in nude mice is significantly more responsive to cisplatin therapy than control of ACHN cell tumors. This work proposes PAX2 as a potential therapeutic gene target in metastatic renal cell carcinoma and suggests that adjunctive PAX2 knockdown may enhance the efficacy of chemotherapeutic agents such as cisplatin. / Wilms tumor, the most common pediatric renal cancer, is thought to arise from a progenitor cell of the metanephric mesenchyme that fails to complete nephrogenesis. In addition to its characteristic triphasic histology, WT can exhibit myogenic differentiation. Myogenic programming during muscle development is controlled by a PAX3 transcription factor determinant for muscle development; unexpectedly PAX3 transcriptional activity has been recently identified in the embryonic mouse kidney. These observations led us to hypothesize that PAX3 plays a role during kidney development. Furthermore, we predict that if PAX3 expression is verified during renal development, PAX3 may also be expressed in Wilms tumor with a myogenic component. / I showed that PAX3 is expressed in the metanephric mesenchyme and stromal compartment of the developing mouse kidney. In a panel of 20 Wilms tumors, PAX3 was identified in tumor samples with myogenic histopathology. Furthermore, mutations of WT1 were consistently associated with PAX3 expression in Wilms tumors and modulation of WT1 expression in HEK293 cells was inversely correlated with the level of endogenous PAX3 protein. / This work supports a novel model of normal renal development in which progenitor cells of the metanephric blastema express PAX3 when targeted toward the stromal cell fate. Suppression of PAX3 is integral to the mesenchyme-to-epithelium transition, which defines the nephrogenic cell fate and may be accomplished, in part, by WT1. Conversely, failure to suppress PAX3 may account for the myogenic phenotype in a subset of WT1-negative Wilms tumors.

Le cancer de l’ovaire est un cancer ayant un taux de décès particulièrement élevé. Les patientes répondent habituellement bien aux traitements chimiothérapeutiques mais la majorité connaîtront une rechute. Plusieurs mécanismes ont été identifiés comme partiellement responsables du développement de la résistance clinique à la chimiothérapie, dont la réparation plus efficace de l’ADN par la réparation par excision de nucléotides (NER). L'un des agents communément utilisés pour traiter ce cancer est le cisplatine, qui induit des dommages à l'ADN réparés par le NER. Une étude précédente de notre laboratoire a démontré qu'une déficience uniquement en phase S de la réparation par le NER peut se produire. Cette déficience est aussi dépendante de la kinase ATR. Nous avons choisi de déterminer si cette déficience est présente dans certains cas de cancer de l’ovaire et si cette déficience joue un rôle sur la résistance à la chimiothérapie. Nos objectifs sont donc : (i) vérifier la présence de cette déficience dans diverses lignées isolées du cancer de l’ovaire ; (ii) vérifier si le traitement chimiothérapeutique par des agents à base de platine peut favoriser la survie de cellules ayant une meilleure capacité de réparation par le NER; (iii) mesurer la sensibilité de ces lignées au cisplatine et vérifier si ceci corrèle avec leur capacité de réparation par le NER en phase S; (iv) déterminer si cette déficience est causée par la kinase ATR dans ces lignées. Nous avons déterminé qu’une déficience importante de la réparation par le GG-NER en phase S est présente dans de nombreuses lignées. De plus, des lignées isolées d’une même patiente pré-chimiothérapie et post-chimiothérapie montrent une augmentation significative de leur capacité de réparation par le GG-NER en phase S, suggérant un rôle de ce processus dans la résistance à la chimiothérapie. Nous avons aussi démontré qu’il y a une corrélation entre la capacité de réparation en phase S par le GG-NER et la sensibilité des lignées au cisplatine. Toutefois, nos résultats suggèrent que cette déficience n’est pas causée par ATR dans ces lignées puisque la phosphorylation de H2AX en réponse aux UV est similaire dans toutes les lignées. En plus d’un important apport fondamental, cette étude permettra d’étudier un potentiel mécanisme de résistance aux traitements chimiothérapeutiques dans le cancer de l’ovaire humain. / Ovarian cancer is one of the most lethal cancers. Patients usually respond very well to chemotherapy but most will eventually relapse. Many molecular processes have already been shown to influence chemotherapy resistance, including more efficient DNA repair by nucleotide excision repair. One commonly used drug for the treatment of ovarian cancer is cisplatin, a drug inducing lesions on DNA that can be repaired by NER. A previous study in our lab has shown that NER can be deficient specifically in S-phase. This effect is dependent on the kinase ATR. Thus, we chose to explore the possibility that this deficiency has an impact on resistance to cisplatin. Our objectives are: (i) to study the repair profile by NER in S-phase in ovarian cancer cell lines; (ii) to test if chemotherapeutic treatment can modulate nucleotide excision repair in cell lines by selecting cells that repair damage by NER more efficiently; (iii) to measure the sensitivity of these cell lines to cisplatin; (iv) to test if this deficiency can be attributed to ATR signalling. We show in this study that many ovarian cancer cell lines have an important defect in GG-NER specifically in S-phase. Pairs of cell lines that were isolated before and after chemotherapeutic treatment show an increase in their GG-NER efficiency in S-phase, suggesting a role for this enzymatic process in chemotherapy resistance. Also, we have found a correlation between the efficiency of GG-NER in S-phase and the sensitivity to cisplatin in the cell lines used in our study. However, the defect in GG-NER in S-phase in these ovarian cancer cell lines doesn’t seem to be due to ATR because the phosphorylation of H2AX in response to UV is equivalent between the different cell lines. This study will have an impact on our understanding of the fundamental aspects of DNA repair but could also provide insights on a potential novel mechanism of resistance to chemotherapy.

In the 1980's, Nicholas Farrell developed a range of structurally distinct multinuclear Pt complexes that form long-range interstrand crosslinks (IXLs) in DNA. The dinuclear complex [{trans-PtCl2(NH3)}2-µ-(H2N(CH2)6NH2)]2+ (1,1/t,t) was the first of this series to show promising results, however, it was the trinuclear complex [{trans-PtCl2(NH3)}2-µ-trans-Pt(NH3)2(H2N(CH2)6NH2)2]4+ (1,0,1/t,t,t or BBR3464) that was chosen for clinical trials based on significantly increased cytotoxicity compared to 1,1/t,t and cisplatin. Molecular biology experiments have shown that 1,1/t,t exclusively forms IXLs in DNA in the 5'¿ 5' direction, whilst 1,0,1/t,t,t can form IXLs in both the 5'¿5' and 3'¿3' directions. Previously, 2D [1H,15N] HSQC NMR has been used to study the formation of 5'–5' 1,4–GG IXLs. The formation of 3'–3' 1,4–GG IXLs have been studied as part of this thesis. More recently, Pt complexes such as [{trans–PtCl2(NH3)}2{H2N(CH2)6(NH2(CH2)2NH2)(CH2)6NH2}]4+ (1,1/t,t–6,2,6) and [{trans–PtCl2(NH3)}2{H2N(CH2)6(NH2)(CH2)6NH2}]3+ (1,1/t,t–6,6), where the charged central Pt moiety of 1,0,1/t,t,t is replaced by a polyamine linker, have been developed in the Farrell group and show increased potency compared to 1,0,1/t,t,t. The complex 1,1/t,t 6,2,6 is a lead candidate currently undergoing Phase I clinical trials. Prior to the work presented in this thesis, little was known about the aquation chemistry or kinetics of DNA binding of these novel complexes. Reported in Chapter 3 is the study of the formation of 3'–3' 1,4–GG IXLs by both 1,0,1/t,t,t and 1,1/t,t in the duplex 5' {d(TATACATGTATA)2} (33–14XL) (pH 5.4, 298K). A combination of 1D 1H and 2D [1H, 15N] HSQC NMR experiments was used to directly compare the results with the stepwise formation of the 5'–5' 1,4–GG IXL with the previously studied duplex, 5' {d(ATATGTACATAT)2} (55–14XL), under the same conditions. Preassociation as well as aquation were similar, however, differences were observed at the monofunctional binding step with evidence for numerous monofunctional adducts. Both reactions did not yield a single 3'–3' 1,4–GG IXL, rather several adducts that could not be characterised. Molecular dynamics simulations of the 3'–3' 1,4–GG IXLs showed highly distorted lesions that may have implication in cellular repair processes.