Tesis sobre el tema "MitoTALEN"

Pendant longtemps, il n'était pas possible de manipuler le génome mitochondrial des plantes (mtDNA), jusqu'aux récentes avancées en édition des génomes utilisant des "Transcription Activator-Like Effector Nucleases" (TALEN). Dans ce travail, j'ai utilisé des TALENs spécifiquement ciblés sur les mitochondries (mitoTALENs) pour étudier la réparation et la ségrégation du mtDNA des plantes. Les constructions de mitoTALEN ont été introduites dans 10 lignées mutantes différentes d'Arabidopsis thaliana, déficientes en divers facteurs impliqués dans la réparation mitochondriale des plantes par recombinaison homologue. Les lignées résultantes ont eté analysées par séquençage Illumina et par des approches de qPCR. Chez les plantes de type sauvage, les cassures double brin (DSB) de l'ADN mitochondrial induites par les mitoTALENs ont été réparées par recombinaison homologue, entraînant le remplacement de la région contenant la DSB par une séquence distale, non-affectée, du mtDNA, flanquée par les mêmes séquences répétées. Chez les mutants déficients en facteurs de réparation, la réparation pourrait se dé placer vers des voies alternatives, telles que le "Single-Strand Annealing" (SSA) et "Microhomology-mediated recombination" (MHMR). De plus, chez certains mutants, les données n'ont révélé aucune trace de réparation des DSB, mais ont plutôt suggéré que les plantes déficientes en facteurs de réparation essentiels pourraient survivre en reconstituant un génome mitochondrial alternatif viable, à partir de sous- génomes pré existants se répliquant de manière autonome
For long, the plant mitochondrial genome (mtDNA) was not amenable to manipulation, until recent advancements in genome engineering using Transcription Activator-Like Effector Nucleases (TALEN). In this work I used TALENs specifically targeted to mitochondria (mitoTALENs) to study plant mtDNA repair and segregation. MitoTALEN constructs were transformed into the background of 10 different Arabidopsis thaliana mutant lines, deficient in various factors involved in plant mitochondrial repair by homologous recombination. The resulting lines were analysed by Illumina sequencing and qPCR approaches. In wild type plants, the mtDNA double-strand-break (DSB) induced by MitoTALENs was repaired by homologous recombination, resulting in the replacement of the region containing the DSB by a distal unaffected sequence of the mtDNA, flanked by the same set of repeated sequences. In mutants deficient in repair factors, repair could shift to alternative pathways, such as Single-Strand Annealing (SSA) and Microhomology-mediated recombination (MHMR). Furthermore, in some mutants, the data revealed no evidence of DSB repair, but rather suggested that plants deficient in key repair factors could survive by reconstituting an alternative viable mitochondrial genome, from pre-existing autonomously replicating sub-genomes

Le mitotane, ou o,p’DDD, médicament historique dérivé d’un insecticide, reste le traitement de référence du corticosurrénalome (CS), tumeur rare et de mauvais pronostic. Le mitotane exerce un effet anti-sécrétoire associé à un effet anti-tumoral dont les mécanismes d’action sous-jacents sont mal compris et ses cibles moléculaires restent inconnues. Néanmoins, de nombreux arguments semblent désigner la mitochondrie comme organite cible du mitotane. Dans ce travail de thèse, nous nous sommes intéressés à l’impact mitochondrial du mitotane par des approches complémentaires sur plusieurs modèles expérimentaux. Nous avons ainsi pu mettre en évidence une inhibition sélective des complexes I et IV de la chaîne respiratoire, une fission du réseau mitochondrial ou encore une activation de la biogenèse mitochondriale. Ces données nous ont permis d’identifier les mitochondrial-associated membranes (MAM) et le transducéosome comme organites cibles du mitotane. Parallèlement, en réévaluant le rôle potentiel du métabolite acide o,p’DDA, nous avons pu exclure son implication dans l’action cytotoxique du mitotane. Nous avons également démontré que le mitotane libre, non lié aux lipoprotéines, était le plus actif in vitro et pouvait ainsi être celui responsable de l’action pharmacologique in vivo. L’ensemble de ces résultats apportent une meilleure compréhension des mécanismes d’action du mitotane et devrait permettre d’identifier des facteurs prédictifs de réponse à ce traitement pour une meilleure prise en charge des patients atteints de CS
Mitotane is o,p’DDD, an historic drug derived from an insecticide and represents the treatment of choice for adrenocortical carcinoma (ACC), a rare adrenal tumor associated with bad prognosis. Mitotane is responsible for an inhibition of steroidogenesis associated with an antitumoral effect but its exact molecular mechanisms of action remain unclear and its molecular target remains unknown. However, mitochondria could be an organite targeted by mitotane. In this study we evaluated mitochondrial impact of mitotane using complementary approaches on several experimental models. We showed a mitotane-induced selective inhibition of respiratory chain complexes I and IV, an increased fission of the mitochondrial network and an activation of the mitochondrial biogenesis. These data helped us to identify mitochondrial-associated membranes (MAM) and so-called transduceosome as targeted organites of mitotane. We also reevaluated the potential role of the acid metabolite o,p’DDA and excluded its implication in the cytotoxic action of mitotane. Finally, we demonstrated that free mitotane which is not linked to lipoproteins is more efficient in vitro and could be therefore responsible for pharmacological action in vivo. Altogether, these results allow to better understand mitotane mechanisms of action and should help to identify predictive markers of response to mitotane for a better care of patients with ACC

Mitotane (o,p_-DDD), a derivative of the insecticide dichlorodiphenyltrichloroethane, has been widely used for treatment of adrenocortical carcinoma (ACC). ACC is a rare neoplasm characterized by a dismal prognosis, with less than 50 % of patients surviving 5 yr after diagnosis. Because the majority of patients undergoing radical resection relapse, adjuvant therapy has been considered as an option. Indeed, it has been shown that adjuvant mitotane treatment has beneficial effects on outcome in ACC patients. Over the years, mitotane therapy has been thoroughly debated because of its toxicity that limited patient compliance and possibly drug efficacy, due to the previously employed high doses without blood concentration monitoring. The important advancement represented by mitotane monitoring allowed to demonstrate that mitotane activity and toxicity is correlated with the achieved blood drug concentrations. However, the undesired effects of mitotane also manifest at concentrations considered to have a therapeutic impact (14–20 mg/liter, corresponding to 44–62 microM), representing a major hurdle to treat adjuvantly patients who are free of disease after radical surgery. Mitotane treatment is associated with several adverse events. Recently, the effects of mitotane on the endocrine system have been thoroughly investigated in patients treated adjunctively after complete ACC removal, where perturbation in thyroid function is characterized by a decrease in FT4 levels over time, inversely correlated with mitotane concentration. Free T3 (FT3) and TSH levels do not change significantly, mimicking central hypothyroidism. Previously, mitotane has been demonstrated to increase T4-binding globulin (TBG) and to compete with T4 for TBG binding sites, but these phenomena do not explain the changes in thyroid function pattern observed during adjuvant therapy. It has been suggested that mitotane might impair pituitary TSH secretion, interfere with FT3, FT4, and/or TSH assays, or affect deiodase activity, thus changing the FT4/FT3 ratio. However, no evidence is currently available to support these hypotheses. Additionally, reduced ACTH levels were observed in patients treated adiuvantly with mitotane. The aim of our study was to investigate whether mitotane may interfere with thyroid hormone and TSH assays in human serum or directly affect TSH secretion in a mouse model of thyrotrope cells. Additionally, the aim of this study was to evaluate mitotane effects on adrenocorticotrope function and ACTH secretion in a mouse model of adrenocorticotrope cells, in order to explain the perturbation of ACTH levels in patients treated adiuvantly with mitotane. Our results demonstrate that mitotane does not interfere with thyroid hormone laboratory tests but directly reduces both secretory activity and cell viability on pituitary TSH secreting mouse cells. These data represent a possible explanation of the biochemical picture consistent with central hypothyroidism in patients undergoing mitotane therapy and open new perspectives on the direct pituitary effects of this drug. Moreover, mitotane inhibits adrenocorticotrope cell viability, inducing apoptotic mechanisms, and reduces ACTH secretion, both basally and after CRH stimulation. These results suggest that mitotane profoundly affect pituitary function, probably without cell specificity.

Le mitotane ou o,p'-DDD est un dérivé organochloré très peu soluble dans l'eau. Il est utilisé dans le traitement du cancer corticosurrénalien métastasé ou inopérable (Lysodren®). En thérapeutique, il est nécessaire d'utiliser de fortes doses pour atteindre généralement au bout de trois mois, la mitotanémie efficace. Cela entraine le plus souvent, des effets indésirables gastroduodénaux et neuromusculaires, qui rendent les patients très peu compliants.L'objectif principal de cette thèse est de mettre au point, d'évaluer et de comparer les différentes formulations de mitotane afin d'améliorer la biodisponibilité du mitotane par rapport à la forme conventionnelle Lysodren®. Dans cette optique, le but recherché en clinique humaine serait de concevoir une forme galénique pouvant permettre d'utiliser de faibles doses journalières afin d'éviter les effets indésirables liés à la toxicité cumulative du mitotane. Pour cela, dans le but d'augmenter sa solubilité et de le rendre plus biodisponible, nous avons encapsulé le mitotane sous formes de particules polymériques et de microémulsions.Nous avons préparé des nanocapsules à partir de polymères biodégradables (la poly-epsilon-caprolactone) (PCL) et d'une association de PCL et de polymères non biodégradables (L'Eudragit® RL). Nous avons également préparé des microparticules de PCL et des systèmes autoémulsionnants ou SMEDDS.L'évaluation des caractéristiques physico-chimiques des particules montre des diamètres de 300 nm pour les nanocapsules et pour les microparticules, des diamètres variant de 40 à 76 µm. Le potentiel zêta est négatif pour les particules de PCL et positif pour celles associant les polymères PCL et RL. Pour les microémulsions, les diagrammes pseudoternaires ont permis le choix d'une association comportant du Capryol®, Tween® 20 et Crémophor® EL (33, 33, 33%). Les microémulsions ont un diamètre d'environ 40 nm. Les profils de libération in vitro du mitotane montrent une cinétique rapide et une quantité de mitotane libérée plus importante pour les microémulsions et les formes particulaires par rapport à la forme conventionnelle Lysodren®.De même, la réalisation de la pharmacocinétique à dose unique de 100 mg/kg chez des lapins montre des biodisponibilités relatives de 339% plus importantes pour les microémulsions, 195% pour les nanocapsules et 187% pour les microparticules. La quantification du mitotane absorbé dans des modèles Caco-2 montre une absorption complète du mitotane au bout de 4h lorsque le mitotane est formulé sous forme de microémulsions. Pour les microparticules et les nanocapsules, 50 et 45% de la dose initiale ont été respectivement absorbées par les cellules Caco-2. Cette évaluation sur le modèle Caco-2 a également confirmé le faible taux de passage de la poudre de mitotane (10%). Enfin, la réalisation des études de passage sur des coupes de jéjunum de rat en chambre de Ussing confirme que la quantité de mitotane qui a diffusé à travers la membrane jéjunale à partir des microémulsions est 5 fois supérieure à celle obtenue à partir de la poudre de mitotane.En conclusion, les microémulsions présentent un intérêt comme forme orale pour améliorer la biodisponibilité du mitotane. Elles ont pour avantage de multiplier la biodisponibilité par un facteur 3 chez le lapin et sont de fabrication peu coûteuse. Elles constituent une réelle alternative à la forme conventionnelle Lysodren® disponible actuellement sur le marché européen
Mitotane or o, p'-DDD is a organochlorine drug, very slightly soluble in water. It is used in the treatment of non resectable and metastasized adrenocortical carcinoma (Lysodren®). In therapy, to achieve therapeutic plasma level, high cumulative doses of mitotane were usually used during 3-5 months. This regimen causes gastrointestinal and neuromuscular side effects and make patients to be less compliants.The main objective of this work is to developp differents formulations of mitotane in order to improve the relative bioavailability when compared with conventional form Lysodren®. To shorten this equilibration time and reduce side effects, it's necessary to develop a new formulation. In order to increase mitotane solubility and make it more bioavailable, we encapsulated mitotane in polymeric particles and microemulsions.We prepared nanocapsules with biodegradable polymers (poly-epsilon-caprolactone) (PCL) and an association of PCL and non-biodegradable polymers (Eudragit®RL). We have also prepared PCL microparticles and a Self Microemulsifying Drug Delivery System or SMEDDS.Nanocapsules and microparticles diameters were respectively 300 nm and 40 to 76 µm. The zeta potential is negative for PCL particles wheras particles combining PCL and Eudragit®RL polymers exhibited positive zêta potential. For microemulsions, we investigated by constructing ternary phase diagrams and choosing the optimal formulation consisted of a mixture of Capryol®, Tween® 20 and Cremophor® EL (33, 33, 33%) with an emulsion diameter of 40 nm. The release of mitotane from SMEDDS and particles was higher and faster than from the conventional form Lysodren®.Pharmacokinetics after single-dose of oral mitotane formulations (100 mg/kg) in rabbits showed a 339% increase of relative bioavailability with microemulsions, 195% with the nanocapsules and 187% with the microparticles. Caco-2 cell culture showed a complete absorption of mitotane after 4h with microemulsions. For microparticles and nanocapsules, 50 and 45% of the initial dose, were respectively absorbed by Caco-2 cells. Caco-2 cells evaluation confirmed the low absorption of the mitotane powder (10%). Finally, Ussing chamber showed that microemulsions pass through the intestinal barrier 5 times higher than a solution of mitotane. In conclusion, microemulsions showed improvement of bioavailability of mitotane by a factor 3 in rabbits and could allow cost effective production. Microemulsions are a real alternative to Lysodren® which is currently available on the European market

Este relatório foi realizado no âmbito do estágio do MIMV da Universidade de Évora que decorreu no Hospital Veterinário da Associação Zoófila Portuguesa. A primeira parte é relativa à casuística acompanhada durante o estágio. A área da clínica médica onde foram observadas mais ocorrências foi a gastroenterologia. A segunda parte aborda uma revisão bibliográfica do tema “hiperadrenocorticismo canino” sendo complementada com um caso clíni-co que foi acompanhado durante o estágio. O hiperadrenocorticismo é provocado pela produção ou administração excessiva de gluco-corticoides. Os sinais clínicos incluem polidipsia, poliúria, polifagia, alopécia simétrica bilateral, abdómen pendular e hepatomegália. Esta síndrome pode ser iatrogénica ou espontânea. O diagnóstico é muito importante para diferenciar a causa de hiperadrenocorticismo, que é es-sencial para o tratamento. Este pode ser cirúrgico ou médico, sendo que os fármacos mais utilizados são o trilostano e o mitotano; Abstract: Clinic and surgery of small animals This report was carried out as part of the curricular traineeship at the Hospital Veter-inário da Associação Zoófila Portuguesa. The first part is related to the clinical cases followed during the training. The area of the medical clinic where most occurrences were observed was gastroenterology. In the second part a bibliographic review is made of the theme "canine hyper-adrenocorticism", complemented with a clinical case that was followed during the referred train-ing. Hyperadrenocorticism is caused by the excessive production or administration of gluco-corticoids. The clinical signs include polydipsia, polyuria, polyphagia, symmetrical bilateral alo-pecia, pot-bellied appearance and hepatomegaly. This syndrome can be iatrogenic or sponta-neous. Diagnosis is very important to differentiate a cause of hyperadrenocorticism, which is essential for treatment. The treatment can be surgical or medical, and the most used drugs are trilostane and mitotane.

Orientador: Cesar Costapinto Santana
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica
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Resumo: O mitotano (o,p?-diclorodifenildicloroetano) é um fármaco utilizado no tratamento de carcinoma adrenocortical. Ele é comercializado na forma racêmica, ou seja, na proporção 1:1 dos seus enantiômeros R e S. A influência da quiralidade da molécula sobre seu efeito farmacológico ainda não foi estudada. Portanto, a separação dos enantiômeros é importante para testes biológicos comparativos de efeitos colaterais. Este trabalho foi desenvolvido com o intuito de estudar a separação deste fármaco pela técnica de cromatografia líquida de alta eficiência utilizando coluna recheada com a fase estacionária quiral O,O?-bis[4-tercbutilbenzoil]-N,N?-dialil-L-tartardiamida. Diferentes combinações de fase móvel foram testadas e os melhores resultados foram obtidos com hexano/acetato de etila na proporção 95/5 (v/v). Experimentos de pulsos com soluções diluídas do traçador e dos enantiômeros do mitotano foram realizados variando a vazão de fase móvel e a temperatura do sistema. Foram determinados as porosidades do sistema, os parâmetros cromatográficos, os dados de equilíbrio, coeficientes de dispersão axial e parâmetros de transferência de massa. Os resultados mostraram separação satisfatória, com número de pratos superando 9000 e fatores de separação na ordem de 1,13. Os valores dos coeficientes de Henry foram maiores que a unidade para ambos os enantiômeros, sendo que o enantiômero mais retido R-(+)-mitotano, apresentou maior afinidade pela coluna quiral. Valores de km superiores a 300 min-1 revelaram baixo efeito dos fenômenos de transferência de massa e consequentemente predomínio dos efeitos termodinâmicos (energia entálpica superior -10 kJ/mol para os enantiômeros). Experimentos a altas concentrações foram realizados com a finalidade de se determinar às isotermas pelo método da análise frontal e também os cromatogramas sob estas condições. Para a concentração da mistura até 16 g/L as isotermas mostraram um bom ajuste ao modelo de Langmuir. A partir da separação em batelada determinaram-se as regiões de separação dos enantiômeros para um sistema cromatográfico contínuo do tipo leito móvel simulado para diferentes concentrações de alimentação da mistura racêmica. Avaliaram-se as variáveis desempenho (consumo de solvente e produtividade) no sistema contínuo e compararam-se com as obtidas em separações em batelada. Melhores resultados foram obtidos para um sistema contínuo do tipo leito móvel simulado
Abstract: Mitotane (o, p'-dichlorodiphenyldichloroethane) is a drug used in the treatment of adrenocortical carcinoma. It is marketed in the racemic form, proportion 1:1 of their R and S enantiomers. The influence of the molecule quirality on its pharmacology effect was not studied yet. For this reason, this separation is important for comparative biological tests of collateral effects. This work was developed with intention to study the separation of this drug via liquid chromatography using columns packed with the chiral stationary phase, O,O?-bis[4-terc-butylbenzoyl] - N, N' - diallyl-L-tartardiamide. Different combinations of mobile phase was tested and better results were achieved with hexane/ethyl acetate in ratio 95/5 (v/v). Pulses experiments with diluted solutions of the inert and the enantiomers were accomplished at different flow rates and temperature. The system porosities, chromatographic parameters, equilibrium constants, axial dispersion and mass transference parameters were obtained. The results showed satisfactory separation, with number of plates overcoming 9000 and separation factors in the order of 1,13. The values of Henry coefficients were greater than one for both enantiomers, the most retained was R (+) -mitotane, and it presented greater affinity for the chiral column. The overall mass transfer coefficient achieved values higher than 300 min-1, demonstrating low mass transfer effect rates and consequently a prevalence of the thermodynamic effects (enthalpy energy greater than -10 kJ/mol for the enantiomers). Experiments in overload conditions were realized in order to determining the isotherms using the method of the frontal analysis as well the chromatograms under these conditions. For the concentration of the mixture smaller than 16 g/L the isotherms showed a good adjusted to the Langmuir model. The separation regions for a chromatographic continuous system like simulated moving bed were determined with different feed concentrations. This permits the comparison of the performance parameters using the continuous system and batch ones
Mestrado
Desenvolvimento de Processos Biotecnologicos
Mestre em Engenharia Química

The DDT metabolite 3-methylsulfonyl-DDE (3-MeSO2-DDE) is bioactivated by cytochrome P450 11B1 (CYP11B1) in the adrenal cortex of mice and forms irreversibly bound protein adducts, reduces glucocorticoid secretion, and induces cell death selectively in cortisol-producing adrenocortical cells. 3-MeSO2-DDE has therefore been proposed as a lead compound for an improved adrenocortical carcinoma (ACC) therapy. The aims of this thesis were to (1) develop in vitro test systems based on murine and human adrenocortical cell lines and to (2) investigate the mechanisms behind 3-MeSO2-DDE toxicity in adrenocortical cells. The cytotoxic and endocrine-modulating effects of 3-MeSO2-DDE were compared to those of o,p′-DDD (mitotane), the current ACC therapy, and to those of several structurally analogous compounds in both murine and human cell lines. 3-MeSO2-DDE bioactivation and cytotoxicity proceeded in a similar manner in the murine adrenocortical Y-1 cell line as in mice in vivo. The effects were highly structure-specific. Moreover, 3-MeSO2-DDE formed irreversibly bound protein adducts and caused cell death also in the human H295R cell line, and was slightly more cytotoxic than o,p′-DDD. However, 3-MeSO2-DDE toxicity in human cells was not affected by the CYP11B1 inhibitor etomidate, suggesting that bioactivation in human cells is performed by additional/other enzyme(s) than CYP11B1. 3-MeSO2-DDE generated biphasic responses in cortisol and aldosterone secretion and in expression levels of the steroidogenic genes CYP11B1, CYP11B2, and StAR. Such hormesis-like responses were not seen for o,p′-DDD or the precursor DDT metabolite p,p′-DDE. In addition, the two o,p′-DDD enantiomers (R)-(+)-o,p′-DDD and (S)-(-)-o,p′-DDD exhibited slight differences in cytotoxic and endocrine-modulating activity in H295R cells. In conclusion, this thesis  provides  extended  knowledge  on  the  mechanisms  of  action  of 3-MeSO2-DDE and points out important differences in effects between murine and human cells. Lead optimisation studies of 3-MeSO2-DDE using the herein presented in vitro test systems are ongoing.

Das Nebennierenrindenkarzinom ist eine hochmaligne Erkrankung und hat eine schlechte Prognose. Mitotane ist bis heute die einzige hierfür zugelassene Therapie. Um die molekularen Mechanismen der Mitotanetherapie besser zu verstehen, wurde die Nebennierenkarzinom-Zelllinie NCI-H295 mit unterschiedlichen Konzentrationen von Mitotane inkubiert und die Wirkung auf mehreren Ebenen untersucht. Dabei kam der Untersuchung der Steroidogenese und apoptotischer Vorgänge ein besonderer Fokus zu. In den Hormonanalysen via Immunoassay zeigte sich eine zeit- und konzentrationsabhängige Hemmung der adrenalen Steroidsekretion. So kam es unter 24-stündiger Inkubation mit 100µM Mitotane zu einer Reduktion der Cortisolsekretion um 89%. Diese Hormonsuppression geht einher mit einer Herabregulation von steroidogenen Enzymen in den durchgeführten Microarray-basierten Genexpressionsanalysen. So konnte gezeigt werden, dass vor allem Steroidbiosynthese-Enzyme der Zona fasciculata und reticularis betroffen sind. Als weitere wichtige Gene im Zusammenhang mit der Beeinflussung des Steroidhaushalts unter Mitotanetherapie konnten SQLE, LDLR, SCD, SREBF1 und ABCG1 identifiziert werden. Gleichzeitig konnte durch Durchflusszytometrie und Zelltod-ELISA die proapoptotische Wirkung von Mitotane gezeigt werden (FACS: 100µM Mitotane, 24 Stunden; Zunahme der Apoptose um den Faktor 2,13). Dies bestätigte sich beispielsweise auch in der Überexpression des Apoptosegens BAX in der Real-Time-PCR. Weiterhin zeigte der RNA-Microarray eine starke Expressionszunahme bei Genen, die mit dem programmierten Zelltod zusammenhängen wie GDF15, DUSP4, TRIB3 und CHOP. Ausgehend von den klinischen Effekten und bestätigt durch die oben genannten in vitro Ergebnisse bewirkt Mitotane auch molekular folgende Änderungen in Nebennierenrindenzellen: Hemmung der Steroidogenese und Induktion von Apotose. Es stellt sich damit die Frage, ob diese Mechanismen parallel und separat voneinander ablaufen oder ob es einen gemeinsamen Nenner gibt. Interessanterweise ergab die Analyse der Genexpressionsdaten, dass viele der proapoptotischen Gene mit dem sogenannten ER-Stress zusammenhängen. Einerseits könnte Mitotane durch direkte Inhibition der Hormonsekretion wirken, andererseits könnte ER-Stress durch Mitotane-induzierte-Bildung toxischer Lipide, wie Cholesterol, ausgelöst werden. Um den genauen Wirkmechanismus endgültig zu klären, werden weitere Experimente benötigt. Mitotane-induzierter ER-Stress liefert einen vollständig neuen Blickwinkel auf die molekulare Wirkweise von Mitotane auf Nebennierenrindenkarzinomzellen. Gerade da die Mediatoren des ER-Stresses gut definiert und ER-Stress spezifisch sind, könnten sie sinnvolle Ziele in der Therapie darstellen. Die Beobachtung, dass Mitotane ER-Stress hervorruft, könnte in Zukunft somit zur Entwicklung wirksamerer und spezifischerer Therapien des Nebennierenrindenkarzinoms führen und so die infauste Prognose dieser malignen Krankheit verbessern
In-vitro studies to elucidate the moclecuar actions of Mitotane in adrenocortical carcinoma

Il carcinoma corticosurrenalico (ACC) è un tumore raro e altamente aggressivo che origina nella corteccia surrenalica, con una prognosi infausta dovuta al suo fenotipo maligno e alla mancanza di opzioni terapeutiche efficaci. Il fatto che a tutt’oggi non siano ancora state sviluppate terapie specifiche si deve attribuire alla scarsa conoscenza dei meccanismi patogenetici di questo tipo di tumore. L’unico farmaco attualmente in uso per il trattamento dei pazienti con ACC ad uno stadio avanzato è il mitotane, di cui tuttavia non sono ancora stati studiati i possibili effetti tossici a livello delle cellule surrenali tumorali e i meccanismi coinvolti. Il primo obiettivo di questo lavoro di tesi si è pertanto incentrato sullo studio dei profili di espressione proteica del tumore e della ghiandola surrenale sana, allo scopo di individuare proteine differenzialmente espresse e di conseguenza in grado di discriminare i due gruppi sperimentali. Lo studio è stato condotto mediante l’utilizzo della tecnica di 2D-DIGE (Differential In-gel Electrophoresis), utilizzata per la prima volta per questo tipo di tumore, che ci ha permesso di confrontare e quantificare i profili proteici di 19 campioni bioptici (11 tessuti tumorali e 8 tessuti surrenalici sani) corsi su gel differenti, grazie alla presenza di uno standard interno costituito da un pool proteico di tutti i campioni analizzati. La spettrometria di massa associata all’analisi DIGE ha identificato 22 proteine differenzialmente espresse (con un’ average ratio ≤-2 o ≥2 e una significatività statistica p<0.05) tra la condizione patologica e quella normale. La maggior parte delle proteine sono risultate overespresse negli ACC, ad eccezione di una downregolata, la tiosolfato sulfotransferasi. Per confermare la modulazione delle proteine identificate, abbiamo utilizzato due diverse metodiche: Western Blotting e immunoistochimica; la scelta delle proteine da validare si è basata sul ruolo che alcune di esse rivestono in alcune vie di segnalazione coinvolte nella tumorigenesi e nella progressione tumorale, ed in particolare abbiamo confermato l’espressione differenziale di ALDH6A1, TRANSFERRINA, FASCINA1, LAMINA A/C, CAP1 e ADX REDUTTASI (fold icrease+SE di 7.5±1.4, 3.6±1.2, 2.9±0.2, 2.6±2.1, 1.9±1.4, 1.6±0.8, p<0.05, rispettivamente) negli ACC rispetto alle surreni sane. In conclusione, i nostri risultati preliminari hanno identificato un profilo proteomico specifico per gli ACC, che si differenzia da quello dei tessuti surrenalici sani, e, all’interno di esso, abbiamo osservato il coinvolgimento di numerosi enzimi mitocondriali che potrebbero rappresentare dei validi biomarcatori per la diagnosi e lo sviluppo di terapie targetspecifiche, se ulteriormente validati in una coorte più estesa di pazienti. Se da un lato sono ancora in gran parte sconosciuti i meccanismi molecolari alla base dello sviluppo e della progressione del tumore corticosurrenalico, e diventa quindi di fondamentale importanza l’utilizzo di differenti approcci per l’individuazione di nuovi possibili markers molecolari che permettano lo sviluppo di terapie più efficaci, dall’altro lato è altrettanto importante uno studio approfondito del meccanismo di azione del mitotane, che ad oggi rimane l’unico farmaco efficacie utilizzato per il trattamento dei pazienti con ACC allo stadio avanzato. Nonostante venga utilizzato da lungo tempo, infatti, non se ne conoscono i target intracellulari e molecolari, e uno studio approfondito potrebbe essere di aiuto per la scelta di eventuali molecole da somministrare in combinazione con esso per ridurne le dosi e ottenere degli effetti citotossici maggiori e maggiormente specifici. Nella seconda parte di questo lavoro, pertanto, siamo andati ad indagare gli effetti del mitotane a livello della linea cellulare di carcinoma corticosurrenalico, H295R, focalizzandoci sullo studio dei meccanismi intracellulari alla base del suo effetto tossico: in particolare, siamo andati ad analizzare le alterazioni nella morfologia e nella funzionalità dei mitocondri. Dopo aver osservato che il mitotane (DDD) viene metabolizzato e si accumula insieme al metabolita DDE all’interno delle cellule H295R in maniera dose-dipendente, abbiamo effettuato dei saggi di proliferazione che hanno confermato il suo effetto citotossico a livello delle cellule tumorali della corteccia surrenalica; in particolare, l’effetto del farmaco è evidente già a partire dalla dose di 10 micromolare dopo 48h di somministrazione, e raggiunge il suo effetto massimo dopo 7 giorni ad alte dosi (con un’inibizione della crescita tumorale del 92+1.12 % alla dose di 30 micromolare, corrispondente alla dose più bassa della finestra terapeutica del farmaco, dopo 7 giorni e significatività statistica p<0,0001). Successivamente siamo andati a valutare l’interessamento delle strutture intracellulari mediante analisi in microscopia elettronica: abbiamo osservato un’alterazione nella morfologia di questi organelli indotta dal mitotane in maniera dose- e tempo-dipendente; i mitocondri hanno mostrato un progressivo rigonfiamento, in concomitanza con una significativa diminuzione del numero delle creste interne. Dal momento che la riduzione nell’estensione del numero delle creste interne può interferire con la funzionalità mitocondriale, siamo andati a valutare se le alterazioni strutturali si riflettessero anche in un’alterazione della funzionalità di questi organelli. A questo scopo abbiamo valutato il potenziale di membrana in cellule vive in adesione, e abbiamo osservato una depolarizzazione dose-dipendente, che esita in una completa disgregazione degli organelli. Infine, per valutare se la depolarizzazione del potenziale di membrana influenzasse anche la respirazione, siamo andati a valutare il consumo di ossigeno in mitocondri vivi isolati da cellule H295R e abbiamo osservato una riduzione del consumo di ossigeno che sembra principalmente dovuta alla presenza di un danno di membrana (come confermato mediante esperimenti in Western Blotting) piuttosto che ad una specifica alterazione degli enzimi della catena respiratoria. Questi risultati contribuiscono a chiarire il meccanismo di azione del mitotane, mostrando che l’alterazione mitocondriale rappresenta uno dei principali bersagli dell’azione citotossica del farmaco. In conclusione, con questo lavoro sperimentale di tesi abbiamo individuato, mediante la tecnica 2D-DIGE, la presenza di un pattern proteico overespresso, specifico dell’ACC, e, all’interno di esso, abbiamo osservato il coinvolgimento di numerosi enzimi mitocondriali, che potrebbero rappresentare dei validi biomarcatori per la diagnosi dell’ACC. Con i dati di questo lavoro di tesi abbiamo quindi evidenziato che il mitocondrio rappresenta uno dei candidati più promettenti per lo sviluppo di nuove terapie target-specifiche, dal momento che anche il mitotane ha dimostrato di svolgere il suo effetto citotossico a livello di questo organello.

博士
國立陽明大學
生理學研究所
100
Adrenocortical carcinoma (ACC) is an extremely rare and aggressive endocrine neoplasm with poor prognosis. Hypercortisolism (Cushing’s syndrome) is the most common complication of functional ACCs. Mitotane not only moderates the signs and symptoms caused by glucocorticoid excess but also improves the metastasis and recurrence of ACC. However, the biological mechanism of anti-steroidogenesis and anti-tumorogenesis of mitotane remains unknown. In this study, adrenocortical carcinoma NCI-H295 cells were used and treated with mitotane (0 - 40 M) for 24 hrs. Mitotane significantly inhibited basal, ACTH (1, 100 nM)-, FK (1, 10 M)- and 8-Br-cAMP (10, 100 M)-induced cortisol secretion in a dose-dependent manner but did not cause cell death. Gene transcription activity of various steroidogenic enzymes including StAR, CYP11A1, CYP11B1, CYP11B2, CYP17 and CYP21 were also diminished by mitotane treatment but not HSD3B2. Interestingly, mitotane expressed biphasic effect on CYP11B1 and CYP11B2 genes. Administration of mitotane potentiated ERK phosphorylation in a dose-dependent manner under both microenvironments. The total protein of SF-1 and DAX-1 were unchanged. These results suggested that mitotane suppressed cortisol production of NCI-H295 cells via interfering basal and cAMP-mediated gene transcription of steroidogenic enzymes. The ERK-mediated signaling might contribute to this effect through altering the protein-protein interaction between SF-1 and DAX-1. VEGF plays a crucial role in angiogenesis and tumor cell growth and previous studies demonstrated that ACC tumor expresses VEGF receptor. Mitotane suppressed basal VEGF production of ACC cells in a dose-dependent manner. Mitotane (40 – 80 M) inhibited VEGF production and cell viability, while 100 M mitotane expressed the more potential inhibitory effect on VEGF production than cell viability. Moreover, the VEGF production induced by CoCl2-induced hypoxia was suppressed by 60-70 M mitotane. These results suggested that mitotane inhibited tumorigenesis might be through blocking VEGF/VEGFR autocrine loop and/or inducing cell death in NCI-H295 cells.

abstract: In wild birds, the stress response can inhibit the activity of the innate immune system, which serves as the first line of defense against pathogens. By elucidating the mechanisms which regulate the interaction between stress and innate immunity, researchers may be able to predict when birds experience increased susceptibility to infections and can target specific mediators to mitigate stress-induced suppression of innate immune activity. Such elucidation is especially important for urban birds, such as the House Sparrow (Passer domesticus), because these birds experience higher pathogen prevalence and transmission when compared to birds in rural regions. I investigated the role of corticosterone (CORT) in stress-induced suppression of two measures of innate immune activity (complement- and natural antibody-mediated activity) in male House Sparrows. Corticosterone, the primary avian glucocorticoid, is elevated during the stress response and high levels of this hormone induce effects through the activation of cytosolic and membrane-bound glucocorticoid receptors (GR). My results demonstrate that CORT is necessary and sufficient for stress-induced suppression of complement-mediated activity, and that this relationship is consistent between years. Corticosterone, however, does not inhibit complement-mediated activity through cytosolic GR, and additional research is needed to confirm the involvement of membrane-bound GR. The role of CORT in stress-induced inhibition of natural antibody-mediated activity, however, remains puzzling. Stress-induced elevation of CORT can suppress natural antibody-mediated activity through the activation of cytosolic GR, but the necessity of this mechanism varies inter-annually. In other words, both CORT-dependent and CORT-independent mechanisms may inhibit natural antibody-mediated activity during stress in certain years, but the causes of this inter-annual variation are not known. Previous studies have indicated that changes in the pathogen environment or food availability can alter regulation of innate immunity, but further research is needed to test these hypotheses. Overall, my dissertation demonstrates that stress inhibits innate immunity through several mechanisms, but environmental pressures may influence this inhibitory relationship.
Dissertation/Thesis
Doctoral Dissertation Biology 2017