Deux complexes de gallium solubles dans l'eau de formule [Ga(III)LCl], où L est la forme déprotonée de dérivés d'acide N-2-hydroxybenzyl-aspartique ont été synthétisés et caractérisés par RMN 1H et 13C, FT-IR, spectrométrie de masse et analyse élémentaire. Les données analytiques obtenues sont cohérentes avec une structure mononucléaire dans laquelle le cation gallium (III) est ligandé par l'un des deux groupes acide carboxylique, l'oxygène phénolique et l’atome d'azote du groupe 2-hydroxybenzylamino. Dans une telle structure, le ligand tridendate assure la liaison de l'ion métallique tandis que l'appendice carboxylique fournit la solubilité dans l'eau. La cytotoxicité du complexe gallium de l'acide (R)-2-(5-chloro-2-hydroxybenzylamino)succinique (GS2) a été évaluée contre les lignées cellulaires de cancer du sein humain MDA-MB231 et de fibrosarcome HT-1080. Nous avons établi que GS2 est plus cytotoxique que le dérivé dépourvu de chlore aromatique et que le chlorure de gallium. GS2 est capable d'induire l'apoptose par la régulation négative de la phosphorylation de l'AKT et l’arrêt du cycle cellulaire en G2M via l’activation de la voie des caspases 3/7. Bien que de nombreux effets moléculaires et cellulaires du Ga aient été décrits, y compris l'inhibition du protéasome et les activités ostéoclastiques, le complexe GS2 apparaît comme le premier complexe de gallium capable de réduire la phosphorylation d'AKT dans les cellules cancéreuses. L'activité de GS2 sur l'invasion cellulaire et sur l'expression et l'activité des métalloprotéinases matricielles (MMP) a été étudiée en utilisant une chambre de Boyden revêtue de collagène de type I. Nous avons analysé l'activité sur les MMPs par zymographie et dosage enzymatique en utilisant des substrats fluorogènes à haute affinité. Une inhibition sélective de MMP-14 a été observée pour bloquer la migration et l'invasion de cellules tumorales. L'expression de l'ARNm des MMP a été analysée par qRT-PCR. GS2 induit une diminution de l'invasion cellulaire. Un effet d'inhibition dose-dépendante a été observé sur les activités de MMP-2, MMP-9 et MMP-14. Une diminution de l'expression de l'ARNm de MMP-14 a été observée dans les deux lignées cellulaires, tandis que l'expression des ARNm de MMP-2 et de MMP-9 ne décroit que dans les cellules tumorales MDA-MB231. Les données obtenues sur l'expression de l'ARNm de MMP-14 ont été confirmées par analyse Western Blot. GS2 semble être une molécule capable de réduire l'activité de MMP-14 dans des maladies métastatiques cancéreuses présentant un niveau élevé d'expression et d'activité de MMP-14. En conclusion, ces données montrent que le complexe GS2, en combinaison avec une chimiothérapie cytotoxique, est un composé prometteur pour la thérapie anti-invasive et anticancéreuse. / Two water soluble gallium complexes with formula [Ga(III)LCl], where L is the deprotonated form of N-2-hydroxybenzyl aspartic acid derivatives were synthesized and characterized by 1H NMR, 13C NMR, FT-IR, mass spectrometry and elemental analysis. The analytical data are consistent with a mononuclear structure in which the gallium (III) cation is liganded by one of the two carboxylic acid groups, the phenol oxygen and the nitrogen atom of the 2-hydroxybenzylamino group. In such a structure, the tridendate ligand secures the binding of the metal ion whereas the carboxylic appendage provides the water solubility. The cytotoxicity of the gallium complex of (R)-2-(5-chloro-2-hydroxybenzylamino) succinic acid (GS2) was evaluated against human breast carcinoma MDA-MB231 and fibrosarcoma HT-1080 cell lines. The 5-chloro derivative GS2 was found to be more cytotoxic than the unsubstituted derivative and GaCl3. GS2 induces apoptosis through down-regulation of AKT phosphorylation, G2M arrest in cell cycle via activation of the caspase3/7 pathway. Although, many molecular and cell effects of Ga have been described, including proteasome inhibition and osteoclastic activities, GS2 appears as the first Ga compounds able to decrease AKT phosphorylation in cancer cells. The activity of GS2 on cell invasion and on the expression and activity of Matrix Metalloproteinases (MMPs) have been investigated using modified Boyden chamber coated with type I collagen. We analyzed the activity on MMPs by zymography and enzymatic assay using high affinity fluorogenic substrates. A selective inhibition of MMP-14 has been reported to blocks tumor cell migration and invasion. The expression of MMPs mRNA was analyzed by qRT-PCR. GS2 induces a decrease in cell invasion. A dose dependent inhibition effect was observed on MMP-2, MMP-9 and MMP-14 activities. A decrease in MMP-14 mRNA expression was observed in both cell lines, whereas MMP-2 and MMP-9 mRNA expression was decreased only in MDA-MB231 cells. Thus, we propose that GS2 compound may be a potential candidate to decrease the MMP-14 activity in cancer metastatic diseases presenting high level of MMP-14 expression and activity. Taken together, these data show that GS2, in combination with cytotoxic chemotherapy a is a promising compound for anti-invasive and anticancer therapy.

Résumé : L’immunothérapie anti-PD1 est à l’origine de résultats cliniques impressionnants dans le traitement de certains cancers comme le mélanome métastatique ou le lymphome Hodgkinien. Néanmoins, les rechutes sont fréquentes et certaines tumeurs y sont d’emblée résistantes. Par ailleurs, l’étude du complexe d’initiation de la traduction eIF4F gagne de plus en plus d’intérêt dans le domaine du cancer. En effet, eIF4F joue un rôle fondamental dans la biologie des cancers grâce au contrôle sélectif de la synthèse de protéines impliquées dans le développement tumoral.Dans cette étude, nous montrons que l’inhibition du complexe eIF4F, en plus d’avoir un effet antitumoral directe via l’inhibition de la croissance tumorale in-vitro, a une action indirecte grâce à l’inhibition de l’expression de PD-L1 sous IFN-g, évitant ainsi le blocage des lymphocytes cytotoxiques suite à l’engagement PD-1/PD-L1. Dans un modèle murin de mélanome, nous avons montré une inhibition de la croissance tumorale grâce à l’inhibition de l’expression de PD-L1, uniquement dans des souris immunocompétentes, montrant ainsi le rôle fondamental du système immunitaire. Nous avons ensuite identifié la voie de régulation de PD-L1 par eIF4F via une régulation traductionnelle de l’ARNm de STAT1, principal facteur de transcription de PD-L1 sous IFN-g.Cette étude apporte une nouvelle preuve de l’intérêt des inhibiteurs d’eIF4F dans le cancer en démontrant leur effet immunothérapeutique via l’inhibition de PD-L1, évitant ainsi l’interaction PD-1/PD-L1 qui conduit à l’échappement des tumeurs. Ces résultats ouvrent la voie vers de nouvelles stratégies dans la lutte contre le cancer. / The eukaryotic translation initiation complex eIF4F is subject of an increased interest in the field of cancer. This heterotrimeric complex, comprising the RNA helicase eIF4A, the cap-binding protein eIF4E and the scaffold protein eIF4G, is known to be more abundant and active in tumor cells than non-malignant counterparts.In a previous work, we showed that this complex is implicated in the resistance to melanoma-targeted therapies (Boussemart et al, Nature 2014). Furthermore, it is implicated in the resistance to various chemotherapies. Thus, agents targeting the eIF4F complex appear as promising tools in the field of cancer therapy.On the other hand, immunotherapy, by (re)stimulating and enhancing the host immune system against tumors is giving good clinical results in oncology treatment and appears nowadays as the most promising approach to fight cancer, especially anti-PD1 treatment. Even though immunotherapy has demonstrated remarkable results in curing some established cancers, such as advanced melanoma or Hodgkin’s lymphoma, many tumors relapse or fail to respond. It is thus important to still look for a new strategy enhancing the efficacy of actual treatments. Here, we propose to study the impact of inhibiting the eIF4F complex on the tumor-specific immune response.

Prostate cancer is the second leading cause of cancer death in men in Europe and the US. In the context of previous preclinical experiments and clinical studies there are certain assumptions predicating successful application of immunotherapy in the treatment of patients with prostate cancer. Promising results have been achieved by a combination of different treatment modalities which provide a synergistic antitumor effect. One of these combinatorial options is the use of antitumor vaccines and adoptive T cell transfer. The topic of this thesis is to provide a fresh insight into the past and current trends following the long-term candidate's department program in the field of anti-tumor immunotherapy. The experimental part of this thesis revolves around our own results published in this field. The introductory chapter delivers a basic overview of cellular mechanisms of anti-tumor immunity and the role of individual immune components in these processes. Following chapters are dedicated to current immunotherapeutic approaches with emphasis on the adoptive T cell transfer and implication of this technology in the treatment of prostate cancer. The results section describes the establishment of our protocol for adoptive T cell transfer as well as the protocol for ex vivo enrichment of human T cell...

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The aim of the study was to evaluate the expression of immunosuppressive (FOXP3, NKG2A), and cytotoxic (perforin) or cytotoxic / immunomodulating (IFNγ) T-cell properties representing biomarkers in the peripheral blood CD8h T-cell population of patients with advanced renal cell carcinoma (RCC) or high risk cutaneous melanoma and healthy controls by multicolour flow cytometry. Determination of the percentage of functionally competing T-cell subsets (especially immunosuppressive) in the CD8hCD57+ T-cell subpopulation in future may serve as one of parameters enabling to assess the overall status of antitumor immune response and select cancer patients most suitable for antitumor immunotherapy while dismissing those to whom it would be ineffective or even harmful. / Darbo tikslas buvo įvertinti imunosupresines (FOXP3, NKG2A), citotoksines (perforin) bei citotoksines / imunomoduliuojančias (IFNγ) savybes atspindinčių žymenų raiškos skirtumus išplitusiu inkstų vėžiu ar didelės rizikos odos melanoma sergančių pacientų periferinio kraujo CD8h T limfocitų populiacijoje, lyginant su kontroline grupe. Skirtingas T limfocitų savybes atspindinčių žymenų raiška buvo tiriama tėkmės citometrijos būdu. Nustatyta, kad inkstų vėžiu ar odos melanoma sergančių pacientų periferiniame kraujyje Įvairių subpopuliacijų (ypač imunosupresinės) nuošimčio nustatymas CD8hCD57+ T limfocitų populiacijoje ateityje gali būti naudingas klinikinėje praktikoje, individualizuojant priešnavikinę imunoterapiją ir selektyviai parenkant tik tuos pacientus, kuriems imuninės sistemos aktyvinimas sukeltų navikinių ląstelių naikinimą, o ne dar labiau gilintų imunosupresiją.

Darbo tikslas buvo įvertinti imunosupresines (FOXP3, NKG2A), citotoksines (perforin) bei citotoksines / imunomoduliuojančias (IFNγ) savybes atspindinčių žymenų raiškos skirtumus išplitusiu inkstų vėžiu ar didelės rizikos odos melanoma sergančių pacientų periferinio kraujo CD8h T limfocitų populiacijoje, lyginant su kontroline grupe. Skirtingas T limfocitų savybes atspindinčių žymenų raiška buvo tiriama tėkmės citometrijos būdu. Nustatyta, kad inkstų vėžiu ar odos melanoma sergančių pacientų periferiniame kraujyje Įvairių subpopuliacijų (ypač imunosupresinės) nuošimčio nustatymas CD8hCD57+ T limfocitų populiacijoje ateityje gali būti naudingas klinikinėje praktikoje, individualizuojant priešnavikinę imunoterapiją ir selektyviai parenkant tik tuos pacientus, kuriems imuninės sistemos aktyvinimas sukeltų navikinių ląstelių naikinimą, o ne dar labiau gilintų imunosupresiją. / The aim of the study was to evaluate the expression of immunosuppressive (FOXP3, NKG2A), and cytotoxic (perforin) or cytotoxic / immunomodulating (IFNγ) T-cell properties representing biomarkers in the peripheral blood CD8h T-cell population of patients with advanced renal cell carcinoma (RCC) or high risk cutaneous melanoma and healthy controls by multicolour flow cytometry. Determination of the percentage of functionally competing T-cell subsets (especially immunosuppressive) in the CD8hCD57+ T-cell subpopulation in future may serve as one of parameters enabling to assess the overall status of antitumor immune response and select cancer patients most suitable for antitumor immunotherapy while dismissing those to whom it would be ineffective or even harmful.

Development of Non-Traditional Platinum Anticancer Agents: trans-Platinum Planar Amine Compounds and Polynuclear Platinum Compounds By Daniel E. Lee, Ph.D. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. Virginia Commonwealth University, 2015 Major Director: Nicholas P Farrell, Ph.D., Professor, Department of Chemistry Platinum anticancer compounds with cis geometry, similar to cisplatin, have been explored to circumvent the cisplatin resistance; however, they were not considered broadly active in cisplatin cells due to exhibiting similar or same cell death mechanism as cisplatin. Platinum compounds with trans geometry were less studied due to transplatin being clinically inactive; but with few structural modifications, they resulted in unaffected cytotoxic activities in cisplatin resistant cells with structural modification by exhibiting different modes of DNA binding. This research focused on further exploring and establishing structure-activity relationship of two promising non-classical series of platinum compounds with trans-geometry: trans-platinum planar amine (TPA) compounds and noncovalently binding polynuclear platinum compounds (PPC-NC). During this research, further optimizations of the reactivity of TPA compounds were accomplished by modifying the leaving carboxylate groups. The effects of modified reactivity were probed by a systematic combination of chemical and biophysical assays, followed by evaluating their biological effects in cells. To establish the structural-activity relationship of PPC-NCs, Mono-, Di-, Tri-, and Tetraplatin NC with charge of 4+, 6+, 8+, and 10+ were synthesized and evaluated by utilizing biophysical and biological assays. Lastly, a new class of polynuclear platinum compounds, Hybrid-PPCs, were synthesized and evaluated to overcome the pharmacokinetic problems of BBR3464, phase II clinical trial anticancer drug developed previously in our laboratory.

O melanoma maligno é a forma mais agressiva dos tumores cutâneos. Sendo o responsável por mais de 75% das mortes relativas á este tipo de câncer. O principal quimioterápico utilizado no tratamento do melanoma é a dacarbazina (DTIC), entretanto, as taxas de resposta são insatisfatórias. O composto DM-1 é um análogo estrutural da curcumina, e por esta razão possui propriedades biológicas semelhantes, como agente antiproliferativo e próapoptótico. A terapia de captura de nêutrons por boro (BNCT) atua por meio da deposição do isótopo 10Boro nas células tumorais e após a irradiação de nêutrons térmicos há produção de partículas alfa e lítio que destroem a célula. Neste trabalho estudou-se o mecanismo de ação destas três terapias, DTIC, DM-1 e BNCT no tratamento do melanoma e seus efeitos em células normais in vitro com a finalidade de obtenção de modalidades terapêuticas diferentes para o tratamento desta neoplasia. A IC50 foi obtida pela metodologia de MTT, além da análise da progressão do ciclo celular e marcadores de morte celular por citometria de fluxo. O composto DM-1 e a BNCT apresentaram efeito citotóxico seletivo para as linhagens de melanoma, com alta produção de radicais livres peroxidados. Nas mesmas condições, estes efeitos foram mínimos em células normais, diferente do tratamento com DTIC. Houve diminuição da proporção de matriz extracelular e colágeno solúvel sintetizado em células de melanoma tratadas com DM-1, BNCT e DTIC, entretanto, o quimioterápico ocasionou isoladamente diminuição também em células normais. O potencial elétrico mitocondrial das células de melanoma foi diminuído nos três protocolos de tratamento, assim como houve aumento na quantidade de DNA fragmentado. Este efeito não foi encontrado em células normais tratadas com DM-1 e BNCT. O composto DM-1 foi capaz de induzir apoptose via intrínseca e extrínseca, avaliado pela Anexina V e por marcadores de cinética e de morte celular. A terapia de BNCT induziu apoptose e necrose, indicando que esta terapia atua por diferentes vias em cada linhagem celular. BNCT e DM-1 induziram aumento na expressão dos marcados próapoptóticos, como Bax, citocromo c, caspase 3 e 8 clivadas, além de diminuir os valores na expressão de ciclina D1 e Ki-67, relacionados com a progressão do ciclo celular e proliferação. O quimioterápico DTIC apresentou alguns indícios de apoptose em células de melanoma, mas seus efeitos em células normais foram extensivos, ocasionando morte e parada do ciclo celular em melanócitos, células endoteliais e fibroblastos. O composto DM-1 apresentou formação de corpos apoptóticos, modificações no citoesqueleto e clivagem de caspase 9 e Parp em linhagens de melanoma humano. Desta forma, o composto DM-1 e a BNCT mostraram-se ferramentas terapêuticas mais eficazes no controle da progressão e no aumento da morte celular em células de melanoma. O poder efetivo da terapia de BNCT e do composto DM-1 faz com que a possibilidade de terapias combinatórias tenha resultados extremamente favoráveis na modulação da resposta proliferativa desses tumores. / Malignant melanoma is the most aggressive skin cancer. It is responsible for more than 75% of deaths. The main and most active chemotherapy in the melanoma treatment is represented by dacarbazine (DTIC), however, response rates are disappointing. The DM-1 compound is a curcumin structural analogue and it has similar biological properties, such as an antiproliferative and pro-apoptotic agent. Boron Neutron Capture Therapy (BNCT) works through the deposition of the isotope 10Boron in tumor cells, with subsequent irradiation of thermal neutrons, which produce alpha particles and lithium that destroy the cell. In this study, the action mechanism of these three therapies, DTIC, DM-1 and BNCT in the melanoma treatment and its effects in vitro on normal cells were studied in order to obtain different therapeutic modalities for cancer treatment. The IC50 was obtained by MTT method, besides the analysis of cell cycle progression and cell death markers by flow cytometry. The DM-1 and BNCT showed selective cytotoxic in melanoma cell lines, with high of free radicals production. In the same conditions, these effects were minimal in normal cells, unlike the treatment with DTIC. There was a decrease in the proportion of extracellular matrix and soluble collagen synthesized in melanoma cells treated with DM-1, BNCT and DTIC, however, only DTIC also resulted in decreased in normal cells. The mitochondrial electrical potential of melanoma cells was decreased in the three treatment protocols, as there was an increase in the amount of fragmented DNA. This effect was not found in normal cells treated with DM-1 and BNCT. The compound DM-1 was able to induce apoptosis by the intrinsic and extrinsic pathways, as assessed by Annexin V, cell death and kinetic markers. BNCT induced apoptosis and necrosis, indicating that this therapy acts through different pathways in each cell line. DM-1 and BNCT induced an increase of pro-apoptotic markers, such as Bax, cytochrome c, cleaved caspase 3 and 8 expression, and they reduced cyclin D1 and Ki-67, expression related to the progression of the cell cycle and proliferation. The DTIC has shown some signs of apoptosis in melanoma cells, but its effect on normal cells were extensive, causing death and cell cycle arrest in melanocytes, fibroblasts and endothelial cells. The DM-1 showed apoptotic bodies formation, cytoskeleton changes and caspase 9 and Parp cleavage in human melanoma cell lines. Thus, the DM-1 and BNCT showed as therapeutic tools more with high effectiveness in controlling the cell cycle progression and cell death increase in melanoma cells. The effectiveness of BNCT and DM-1 makes the possibility of combinatorial therapies, with extremely favorable results in the modulation of the proliferative response of these tumors.

Estudos sobre a inserção de complexos oxindolimínicos de cobre(II) ou zinco(II) em argilas sintéticas beidelitas foram desenvolvidos visando obter carregadores inorgânicos capazes de promover a liberação modificada de metalofármacos. Investigações anteriores mostraram que os complexos estudados são promissores agentes antitumorais. São capazes de se ligar ao DNA, nas alças menor ou maior dependendo do ligante, causando danos oxidativos através da formação de espécies reativas de oxigênio (EROs), especialmente radicais hidroxil, no caso de complexos de cobre, que apresentam atividade redox. Também interagem com mitocôndrias levando a uma diminuição do potencial de membrana e atuando como agentes desacopladores. Constituem assim compostos indutores de apoptose ou morte celular programada. Adicionalmente inibem proteínas envolvidas no ciclo celular, como topoisomerase I humana e quinases dependentes de ciclina (CDK1 e CDK2). As beidelitas foram sintetizadas e caracterizadas por métodos já descritos na literatura. Posteriormente, em presença dos complexos, formaram materiais híbridos do tipo beidellita/[complexo]. A caracterização das argilas e dos materiais híbridos obtidos foi realizada usando diferentes técnicas: espectroscopias UV/Vis, IV e EPR, análise termogravimétrica, difratometria de raios X. Na caracterização dos complexos utilizou-se ainda espectrometria de massas (ESI-MS). Os materiais híbridos preparados mostraram-se estáveis e capazes de inviabilizar células tumorais (HeLa), no caso dos materiais híbridos com compostos de cobre(II), com IC50 na faixa de 0,11 a 0,41 mg/mL. Ao contrário, os compostos análogos de zinco(II) e as argilas puras mostraram-se não-tóxicas frente às mesmas células. Os resultados obtidos indicam uma promissora possibilidade de uso das beidellitas como carregadores destes complexos metálicos. / Studies on the insertion of oxindolimine complexes of copper(II) or zinc(II) in synthetic beidellites clays have been developed to obtain an inorganic support capable of promoting the modified-release of metallopharmaceuticals. Previous investigations have shown that the studied complexes are promising antitumor agents. They are able to bind to DNA, in the minor or major groves, causing oxidative damage via formation of reactive oxygen species (ROS), especially hydroxyl radicals, in the case of copper complexes, which have redox properties. They also interact with mitochondria leading to a decrease of membrane potential and acting as decoupling agent. Thus, these compounds can induce apoptosis or programmed cell death. Additionally, they inhibit proteins involved in the cell cycle, such as human topoisomerase I and cyclin dependent kinases (CDK1 and CDK2). The beidellites were synthesized and characterized by methods described in the literature. Additionally, in the presence of these complexes were obtained hybrid materials type beidellita/[complex]. The characterization of clays and the obtained hybrid materials were performed using different techniques: spectroscopy UV/Vis, IR and EPR, thermogravimetric analysis, X-ray powder diffraction. In the characterization of the complexes mass spectrometry (ESI-MS) was also used. The prepared hybrid materials were stable and able to derail tumor cells (HeLa) in the case of hybrid materials with copper(II) compounds, with IC50 in the range from 0.11 to 0.41 mg/mL. Unlikely, the analogous compounds of zinc(II) and pure clays have proved to be non-toxic facing the same cells. These results indicate a promising possibility of using the beidellites as carriers of these antitumor metal complexes

A espécie Streptomyces olindensis DAUFPE 5622 produz o complexo antibiótico retamicina, que apresenta atividade contra leucemia em seres humanos mas possui limitações de uso devido ao seu grande efeito cardiotóxico. Uma linhagem mutante de S. olindensis, So20, obtida por tratamento da linhagem selvagem com MMS (metanossulfonato de metila), apresentou diferença qualitativa quanto à produção deste complexo antibiótico. O extrato metanólico das células desta linhagem mutante sofreu diversos tratamentos com solventes orgânicos e foi submetido a diversas técnicas de separação cromatográfica. Algumas substâncias foram parcialmente isoladas e foram submetidas a análise espectrométrica, que indicaram diferenças nas suas estruturas com relação ao complexo obtido da linhagem selvagem DAUFPE 5622. Algumas destas frações parcialmente purificadas da linhagem mutante foram testadas quanto a sua atividade antimicrobiana, contra Bacillus subtilis 007, e quanto sua capacidade de intercalação em fragmentos de DNA em gel de eletroforese, no ICB-USP, indicando resultados promissores. / Streptomyces olindensis DAUFPE 5622 species produces the antibiotic complex retamycin, which are active against human leukemia, but is still not used in human treatments due to its cardiotoxic effects. The S. olindensis mutant, So20, obtained by treatment with MMS (methyl methanesulfonate), showed qualitative differences in the antibiotic production. The methanolic extract from these mutant strains cells was purified by means of solvent partition and by chromatographic purification techniques. Some compounds were partially purified and the analysis of their spectrometric data indicated structural differences from that obtained from DAUFPE 5622 strains. Some of these partially purified substances from mutant strains were tested for their antimicrobian activity, against Bacillus Subtilis 007, and for their DNA intercalation ability on eletroforesis gel, in the ICB-USP laboratories, showing promising results.