L’objectif principal de cette thèse a été le développement, la caractérisation et l’évaluation in vitro et in vivo de différents nanovecteurs plus spécifiquement les nanoparticules spécifiques pour le traitement de la mélanome et β-lapachone-lipossomale incorporée dans des hydrogels de biopolymère pour la cicatrisation de blessures topiques. La première étape de cette thèse a consisté en la révision de la littérature liées aux résentes avancées sur les nanoparticules pour le ciblage d’agents thérapeutiques pour des cellules circulantes et des mésenchymateuses de mélanomes. De plus, cette révision a approfondi la connaissance sur les principaux biomarcateurs qui étaient déjà identifiés dans ces cellules et quelles caractéristiques des nanovecteurs peuvent influencer leur performance in vivo. Dans la phase expérimentale, les nanoparticules ont été développées en utilisant la méthode de nanoprécipitation de polymères dérivés du poli(γ-benzyl-L-glutamate). Ensuite, des immuno-nanoparticules combinées avec l’anticorps MART-1 spécifique pour les cellules de mélanome, ont été obtenues par le lien streptavidine-biotine. La combinaison de l’anticorps sur la superficie des nanoparticules a été évaluée par western blot. Les nanoparticules ont été caractérisées et évaluées in vitro dans des cellules de mélanome B 16-GFP et des cellules endothéliales de la veine ombilicale humaine (HUVECs), l’activation de complément a été recherchée par la technique d’immunoélectrophorèse bidimensionnelle. Les nanoparticules ont présenté des tailles comprises entre 20 et 100 nm et une charge négative (-3 à -30 mV). La combinaison de l’anticorps sur la superficie des nanoparticules a été observée par la technique western blot et confirmée par les altérations de la taille et de la charge de superficie des particules. Les nanoparticules développées n’ont pas été capables d’activer le systhème complémentaire étant considérées de longue circulation sanguine. Pour l’analyse in vitro, les nanoparticules n’ont pas présenté de cytotoxixité lorsqu’elles ont été testées dans des cellules de mélanome ou dans des cellules normales endothéliales. Lors des tests de capture cellulaire, les immuno-nanoparicules, qui contenaient l’anticorps spécifique pour la reconnaissance de l’antigène surexprimé dans des cellules de mélanome, ont présenté une augmentation de 40 à 50% lors de la capture pour ces cellules, cela indiquant une plus grande spécificité de ce nanovecteurs. La deuxième partie de cette thèse a été le développement, la caractérisation et l’évaluation de l’activité cicatrisante in vivo de β-lapacho encapsulé dans des liposomes multilamellaires incorporés dans de l’hydrogel de biopolymère produit par la bactérie Zoogloea sp. (β-lap-Lipo/ZBP/HEC). β-lap-Lipo/ZBP/HEC a présenté un pH et un comportement rhéologique approprié pour l’application topique, ainsi qu’un profil de libération plus lent de la β-lapachone à partir de l’hydrogel. Les analyses hystopotologiques de l’activité cicatrisante in vivo ont montré que le véhicule hydrogel de biopolymère a été capable de stimuler les réparations du tissu, augmenter la cellularité locale, les fibroblastes, les cellules inflamatoires, les vaisseaux sanguins et les fibrilles de collagène pendant la phase proliférative de la cicatrisation. De plus, β-lap-Lipo/ZBP/HEC a favorisé une augmentation de l’angiogenèse locale et une dimminution de l’inflamation de la blessure. Ces résultats ont montré le potentiel de l’application topique de β-lap-Lipo/ZBP/HEC pour la thérapie des lésions. Pour conclure, cette thèse a contribué au développement de nanovecteurs promisseurs ayant différentes applications biologiques et formes d’administration, comme le traitement systémétique de la mélanome et l’action topique lors de la cicatrisation des blessures / This thesis had as general objective the development, characterization and evaluation in vitro or in vivo of different nanocarriers, specifically site-specific nanoparticles for the treatment of melanoma and liposomal-hydrogel containing β-lapachone for topical wound healing. The first part of this thesis consisted in a literature review about the recent advances in nanoparticles for the targeting of therapeutic agents to circulating and mesenchymal melanoma cells. In addition, this review deepened the knowledge about the main biomarkers identified in these cells and which characteristics of nanocarriers may influence on their in vivo performance. In the experimental phase, nanoparticles were developed through the nanoprecipitation method of polymers derived from poly (γ-benzyl-L-glutamate). Next, immunonanoparticles conjugated with MART-1 antibody specific for melanoma cells were obtained through the streptavidin-biotin binding. The conjugation of this antibody on the nanoparticles surface was evaluated by western blot. The nanoparticles were characterized and evaluated in vitro in B16-GFP melanoma cells and human umbilical vein endothelial cells (HUVECs) and the complement activation was investigated by bidimensional immunoelectrophoresis. The nanoparticles presented sizes between 20 and 100 nm and negative surface charge (-3 to -30 mV). The conjugation of antibody on the nanoparticle surfaces was detected by the western blot technique and confirmed by the changes in particle size and surface charge. The developed nanoparticles were not able to activate the complement system being considered long blood circulation. Regarding the in vitro analysis, the particles did not show cytotoxicity when tested in melanoma cells or normal endothelial cells. In the cell capture assays, the immunonanoparticles, containing a specific antibody for the recognition of the overexpressed antigen in melanoma cells, showed an increase of 40 to 50% in the uptake for these cells, indicating a specificity of this nanocarrier. The second part of this thesis consisted of the development, characterization and evaluation of the in vivo wound healing activity of β-lapachone encapsulated in multilamellar liposomes and incorporated in a biopolymer hydrogel produced by Zoogloea sp (β-lap-Lipo/ZBP/HEC). β-lap-Lipo/ZBP/HEC presented pH and rheological behavior suitable for topical application, as well as a slower release profile of β-lapachone through the hydrogel. Histopathological analyzes of the healing activity in vivo, showed that the biopolymer hydrogel vehicle was able to stimulate tissue repair, with the increase of local cellularity, fibroblasts, inflammatory cells, blood vessels and collagen fibers, during the proliferative phase of wound healing. In addition, β-lap-Lipo/ZBP/HEC promoted an increase in local angiogenesis and a decrease of inflammation at the wound site. These results demonstrate a promising topical application of β-lap-Lipo/ZBP/HEC for wound therapy. In conclusion, this thesis contributed for the development of promising nanocarriers with different biological applications and administration routes, such as systemic treatment of melanoma and topical action in wound healing.

<p><strong><em>Background</em></strong>: Chemotherapy and radiation resistance are major causes of failure in cancer treatment. The response to treatment in cancer cells depends on several mechanisms and pathways such as Janus kinases-signal transducers and activators of transcription JAK/STAT pathway. STAT1 was the first described transcription factor in the STAT family. STAT1 is activated by stimulation of signaling proteins such as type II interferon (IFN- γ) and the activated STAT1 translocates from cytoplasm to nucleus. The translocation of STAT1 would result in transcription and changes in the cell activity in terms of apoptosis, proliferation and angiogenesis. Overexpression of STAT1 is suggested to be involved in the development of resistance to chemotherapy and radiation. In this study, we were interested in finding an inhibitor of the STAT1 translocation. <strong><em>Material and methods</em></strong>: The cervix carcinoma cell line, HeLa, was exposed to test compounds for 2h and were then stimulated with IFN-γ to induce the translocation of STAT1. To detect STAT1-protein and the nucleus, the cells were stained with fluorescent antibodies and Hoescht 33324, respectively, using a STAT1 activator assay. The difference in fluorescence intensity between cytoplasm and nucleus was measured using a high-content microscope, ArrayScan^®. <strong><em>Results</em></strong>: β-lapachone and CRA-1 were found to be inhibitors of STAT1 translocation.</p>

Background: Chemotherapy and radiation resistance are major causes of failure in cancer treatment. The response to treatment in cancer cells depends on several mechanisms and pathways such as Janus kinases-signal transducers and activators of transcription JAK/STAT pathway. STAT1 was the first described transcription factor in the STAT family. STAT1 is activated by stimulation of signaling proteins such as type II interferon (IFN- γ) and the activated STAT1 translocates from cytoplasm to nucleus. The translocation of STAT1 would result in transcription and changes in the cell activity in terms of apoptosis, proliferation and angiogenesis. Overexpression of STAT1 is suggested to be involved in the development of resistance to chemotherapy and radiation. In this study, we were interested in finding an inhibitor of the STAT1 translocation. Material and methods: The cervix carcinoma cell line, HeLa, was exposed to test compounds for 2h and were then stimulated with IFN-γ to induce the translocation of STAT1. To detect STAT1-protein and the nucleus, the cells were stained with fluorescent antibodies and Hoescht 33324, respectively, using a STAT1 activator assay. The difference in fluorescence intensity between cytoplasm and nucleus was measured using a high-content microscope, ArrayScan®. Results: β-lapachone and CRA-1 were found to be inhibitors of STAT1 translocation.

Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2017-05-04T17:34:06Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) dissertação-31 05 2016 correções da biblioteca (atualizada).pdf: 2843933 bytes, checksum: 1003152372d1c305370bd514f8eae798 (MD5) / Made available in DSpace on 2017-05-04T17:34:06Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) dissertação-31 05 2016 correções da biblioteca (atualizada).pdf: 2843933 bytes, checksum: 1003152372d1c305370bd514f8eae798 (MD5) Previous issue date: 2016-03-09 / A β-lapachona é uma naftoquinona (3,4-dihidro-2,2-dimetil-2H-naftol[1,2-b]pirano-5,6-diona) de peso molecular 243,3 g.mol-1 presente na Tabebuia avellanedae Lor, árvore conhecida como ipê roxo ou pau d’arco roxo. Esta naftoquinona é uma substância que pode ser obtida por uma semi-síntese a partir do lapachol bem como do lomatiol. Este bioativo apresenta múltiplos efeitos farmacológicos, como antibacteriano, antifúngico, antiviral, anti-inflamatório e anti-proliferativo. Segundo o sistema de classificação biofarmacêutica, a β-lapachona (BL ou β-lap) é classificada como classe II apresentando baixa solubilidade e alta permeabilidade. Desta forma, faz necessário um estudo de incremento de solubilidade aquosa. O objetivo deste trabalho foi caracterizar físico-quimicamente os complexos propostos utilizando dois polímeros: o dendrímero bis-MPA (DEN) e a cucurbit[6]urila (CUC). O presente estudo mostra duas metodologias para a formação de complexos entre a β-lapachona /dendrímero (BLD) e β-lapachona /cucurbiturila (BLC), visando o aumento da solubilidade deste fármaco através de interações intermoleculares e suas respectivas caracterizações físico-químicas. O complexo com o dendrímero foi preparado a partir de uma relação massa/massa de 3(fármaco):1 (carreador) variando o tempo de adsorção de 1, 3 e 7 dias. Para os complexos com a cucurbiturila uma relação molar 1:1 foi estabelecida para a formação dos complexos de inclusão com BL, variando o período de adsorção em 1, 3 e 7 dias. Após o período de agitação, os complexos BLD e BLC foram evaporados e secos a temperatura ambiente para posterior caracterização. Os complexos obtidos foram caracterizados por cromatografia líquida de alta eficiência, ultravioleta na região do visível, espectroscopia de absorção vibracional na região do infravermelho, espectrometria de ressonância magnética nuclear de hidrogênio, análise termogravimétrica, análise calorimétrica diferencial, difratometria de raio-X e microscopia eletrônica de varredura. Para o complexo BLD ficou evidente que a variação no tempo de adsorção não interferiu na quantidade de produto formado ou nas propriedades do complexo obtido. Os resultados das caracterizações mostram que o BLDD1 e o BLCD1 foram formados de acordo com os dados espectroscópicos e espectrométricos que demonstram variações nos sinais de BL quando complexado. Houve uma mudança na cristalinidade da BL, a qual era bastante cristalina, para um material mais amorfo com morfologia distinta do fármaco isolado. Os resultados das caracterizações térmicas exibiram, para os complexos, uma maior estabilidade térmica da β-lap complexada (BLDD e BLCD) e menor energia de fusão, quando comparado com β-lapachona livre. Essas informações também sugerem uma diminuição na cristalinidade do complexo o que é indicativo para o incremento de solubilidade. Desta forma propôs-se a caracterização físico-química como um indício de melhoria na solubilidade da β-lapachona através da formação de complexos entre BL e dois carreadores distintos e suas contribuições para estudos de uma possível formulação farmacêutica futura. / The β-naphthoquinone lapachol is a (3,4-dihydro-2,2-dimethyl-2H-naphthol [1,2-b] pyran-5,6-dione) molecular weight of 243,3g / mol present in Tabebuia avellanedae Lor, tree known as purple ipe or pau d'purple bow. This naphthoquinone is a substance that can be obtained by semi-synthesis from lapachol well as lomatiol. This bioactive has multiple pharmacological effects such as antibacterial, antifungal, antiviral, anti-inflammatory and anti-proliferative. According to biopharmaceutical classification system, the β-lapachone (BL or β-LAP) is classified as class II presenting low solubility and high permeability. Thus, an increase aqueous solubility study is necessary. The aim of this study was to characterize chemically-physical proposed complexes using two polymers: the bis-MPA dendrimer (DEN) and cucurbit[6]uril (CUC). This study shows two methods for the formation of complexes between β-lapachone / dendrimer (BLD) and β-lapachone / cucurbituril (BLC), in order to increase the solubility of the drug through intermolecular interactions and their physicochemical characterizations. The complex with the dendrimer was prepared from a mass / mass 3 (drug): 1 (carrier) varying the time for adsorption of 1, 3 and 7 days. For cucurbituril complexes with a molar ratio of 1: 1 was established for the formation of inclusion complexes with BL varying the adsorption period at 1, 3 and 7 days. After the stirring period, the BLC and BLD complexes were evaporated and dried at room temperature for further characterization. The obtained compounds were characterized by high-performance liquid chromatography, ultraviolet into the visible region spectroscopy, vibrational absorption in the infrared, nuclear magnetic resonance spectrometry of hydrogen, thermogravimetric analysis, differential scanning calorimetry, X-ray diffraction and electron microscopy scanning. For BLD complex it was evident that the variation in the adsorption time not interfere in the amount of complex formed or the product properties obtained. The results of the characterizations show that BLDD1 and BLCD1 were formed in accordance with spectroscopic and spectrometric data showing variations in signals when complexed BL. There was a change in the crystallinity of the BL, which was very clear, to a more amorphous material with distinct morphology of the drug alone. The results showed the thermal characterizations for the complex, greater thermal stability of the complexed β-LAP (BLDD and BLCD) and less fusion energy, compared with β-lapachone free. This information also suggests a decrease in crystallinity of the complex which is an indication to increase solubility. Thus it was proposed to physicochemical characterization as a better indication of the solubility of β-lapachone by forming complexes between BL and two different carriers and their contributions to studies of possible future pharmaceutical formulation.

Quinones have been the subject of much interest due to their various biological activities, mainly as antitumor and as trypanocidal agents. Quinones are cytotoxic by two main mechanisms: the generation of ROS resulting in oxidative stress and alkylation of cellular nucleophiles, such as DNA and some enzymes such as topoisomerases. Their activity depends on bioreduction, similarly to what happens to nitroaromatic compounds. They also catalyze electron transfer reactions in biological processes and, after reduction generate radical anions (semiquinone radical anion and nitro), which depending on stability, can furnish their free electrons to acceptor molecules. In the present study, compounds with mixed functionalities derived from nor-β-lapachone, including a nitroaniline group were electrochemically studied in protic (acetate buffer) and aprotic (DMF+TBABF4, DMSO+TBAP and Acetonitrile+TBABF4) media, using glassy carbon and mercury as working electrodes. The compounds showed a complex redox behavior and the mechanism was elucidated using electron spin resonance. The electroreduced products of nor-β-lapachone and of the nitroquinones reacted with oxygen, indicative of the generation of reactive oxygen species, reactivity in the order of 2 > nor-β-lapachone > 3 > 1. We investigated their interaction with DNA, which was shown to be positive for nitroquinones and negative for the precursor nor-β-lapachone, in agreement with biological assays which had also shown that the nitroquinones cause DNA damage. The stability of the nitrosemiquinones, their half-life times were measured using mercury electrode, and the reaction rates for the electrochemical process-following-up-isproportionation reaction were measured. From these studies, a lower stability for the meta-substituted nitrophenylaniline (k2 = 5.188 x 103 L mol-1 s-1 and t1/2 = 0.06 s) was evidenced. Upon spectroelectrochemical reduction studies of the nitroquinones, the generation of radicalar intermediates (semiquinone radical anion and nitro radical anion) was observed, with differences between o- and m-derived compounds and the p-substituted one. To increase the solubility of the nitroquinones, in order to allow in vivo studies, the formation of inclusion complexes with β-cyclodextrin were evaluated. Positive results were obtained, leading to a viable formulation alternative for further biological studies with the compounds. / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Quinonas despertam muito interesse devido às suas diversas atividades biológicas, principalmente como agentes antitumoral e tripanossomicida. A citotoxicidade de quinonas decorre de dois mecanismos principais: geração de EROs resultando no estresse oxidativo e alquilação de nucleófilos celulares, como o DNA e algumas enzimas, como as topoisomerases. Sua atividade depende de biorredução, similarmente ao que acontece com compostos nitroaromáticos. Eles também catalisam reações de transferência de elétrons biológicas e, após a redução geram ânions radicais (ânions radicais semiquinona e nitro), que dependendo da estabilidade, podem transferir seus elétrons livres a moléculas aceptoras. Neste trabalho, compostos de funcionalidade mista derivados da nor-β-lapachona, com um grupo nitroanilina, foram estudados eletroquimicamente nos meios prótico (tampão acetato) e aprótico (DMF + TBABF4, DMSO + TBAP e acetonitrila + TBABF4), utilizando carbono vítreo e mercúrio como eletrodos de trabalho. Os compostos apresentaram um comportamento redox complexo e o mecanismo elucidado por espectroeletroquímica. Os produtos da eletro-redução da nor-β-lapachona e nitroquinonas reagiram com oxigênio, indicativo da geração de EROs, na ordem de reatividade 2 > nor-β-lapachona > 3 > 1. Foi investigada a interação com DNA, que se mostrou positiva para as nitroquinonas e negativa para o precursor nor-β-lapachona, concordantes com ensaios biológicos que também evidenciou que as nitroquinonas causam dano ao DNA. A estabilidade do nitrosemiquinona, tempos de meia-vida e as taxas reacionais referentes à reação química de desproporcionamento acoplada ao processo eletroquímico para cada derivado foram determinadas usando eletrodo de mercúrio. A partir desses estudos, foi evidenciada a menor estabilidade para o nitrofenilamina meta-substituída (k2 = 15,188 x 103 L mol-1 s-1 and t1/2 = 0,06 s). Nos estudos por espectroeletroquímica das nitroquinonas, observou-se a geração de intermediários radicalares (ânions radicais semiquinona e nitro), com diferenças entre os nitroderivados o- e m-, assim como para o p- derivado. Com objetivo de aumentar a solubilidade das nitroquinonas, para posteriores estudos in vivo, foi avaliada a formação de complexos de inclusão com β-ciclodextrina. Foram obtidos resultados positivos, refletindo em alternativa viável para formulações posteriores com estes compostos e ensaios biológicos.

Cancer is one of the most dreadful diseases due to its figure of morbidity and mortality and its strong social and economic impact in public healthy. The development of new chemotherapics is urgently required. Pharmoelectrochemical studies are important tools in pharmaceutical chemistry, pharmacology and biomedicine. In the present work, the cytotoxic activity of the nitroquinone 2,2–dimethyl–3-(3-nitrophenylamine)-2,3-dihydronaphtho[1,2-b]furan-4,5-dione, was performed, using electrochemical and pharmacological methods, in order to elucidate its mechanism of reduction and biological action. Additionally, electrochemical behaviour of cytotoxic arylamine and [1,2,3] triazolic naphthoquinones was investigated, in order to obtain reduction potentials, to correlate them with their biological activity. Studies were undertaken in aprotic medium (DMSO/TBAP and Acetonitrile/TBABF4) and protic medium (acetate buffer pH 4.5). The nitroquinone was further investigated, concerning its cytotoxicity, genotoxicity, cells morphological modification and immunomodulation effects in its presence, using, for that, dsDNA biosensors and flow cytometry, with the main goal of proposing its pharmacological mechanism of antitumour action. The nitroquinone is not genotoxic and it caused apoptosis, by an intrinsic (mithocondrial) pathway. The electrochemical behaviour of all the quinones, in aprotic and protic media, are typical of well-behaved quinones. The triazole ring worked as a highly electronwithdrawing group, shifting positively all the redox potentials. The nitroquinone has hybrid electrochemical behaviour due to the presence of two eletroactive groups in the molecule. The analysis by ESR allowed the definition of its reduction mechanism. Reduction potential values (EpIc) of the two sets of substances are not directly related to the pharmacological activities of the compounds. Log P values showed better correlation with the biological activity. With the aid of dsDNA biosensor, and ssDNA in a modified electrode, it was possible to analyse the positive and direct interaction of acridines with DNA. The adaptation of the methodology of ssDNA, enabling the study of these (insoluble) substances in ssDNA, demonstrated the contribution of pharmoelectrochemistry to the discovery of the mechanism of action of drugs. / Conselho Nacional de Desenvolvimento Científico e Tecnológico / O câncer é uma das doenças que mais causam temor na sociedade, devido ao seu potencial de morbidade e mortalidade, seu grande impacto econômico em saúde pública e, em todos os setores produtivos. O desenvolvimento de novos fármacos anticâncer é necessário e urgente. Estudos farmacoeletroquímicos constituem ferramentas importantes em química farmacêutica, farmacologia e biomedicina. No presente trabalho, avaliou-se o potencial citotóxico da nitroquinona 2,2–dimetil–3-(3-nitrofenilamino)-2,3-di-hidronafto[1,2-b]furan-4,5-diona, por meio de métodos farmacológicos e eletroquímicos, visando à elucidação de seu mecanismo de redução e de ação citotóxica. Investigou-se, adicionalmente, o comportamento eletroquímico e citotóxico de naftoquinonas [1,2,3]-triazólicas citotóxicas, com o intuito de determinar seus potenciais de redução para avaliar a possibilidade de correlação com a atividade biológica. Foram realizados estudos eletroquímicos em meio aprótico (DMSO/TBAP e Acetonitrila/TBABF4) e prótico (tampão acetato pH 4,5). A nitroquinona foi investigada detalhadamente quanto à citotoxicidade, à genotoxicidade, na avaliação de modificações morfológicas celulares, aspectos de imunomodulação, empregando biossensores de DNA e citometria de fluxo, com o intuito de propor o mecanismo de ação farmacológica da atividade antitumoral. Ela não se mostrou genotóxica e seu mecanismo de ação envolve a via apoptótica, dependente da mitocôndria. Todas as quinonas heterocíclicas apresentam comportamento eletroquímico típico de quinonas bem comportadas, em meio aprótico e prótico. O anel triazólico funcionou como um grupo altamente eletroatraente, deslocando positivamente os processos de redução desses compostos. A nitroquinona possui comportamento eletroquímico híbrido devido à presença de dois grupos eletroativos na molécula. A análise por ESR definiu o mecanismo de redução desta substância. Os potenciais de redução catódicos (EpIc) das duas séries de substâncias não estão diretamente relacionados às atividades farmacológicas dos compostos. Os valores de Log P correlacionam-se melhor com as atividades citotóxicas. As acridinas, estudadas em biossensor de DNA e em presença de ssDNA, mostram interação direta com o DNA. A adaptação da metodologia do biossensor de DNA possibilitou o estudo dessas substâncias (insolúveis) em ssDNA, demonstrando a contribuição da farmacoeletroquímica na descoberta do mecanismo de ação de fármacos.

Les peroxyrédoxines sont des enzymes essentielles de la cellule. Outre leur rôle d’antioxydant, elles sont aussi des régulateurs de la signalisation cellulaire et des suppresseurs de tumeurs. La péroxiredoxine 1 (Prx1) est la plus abondante parmi les six isoformes de peroxyrédoxines humaines. Elle est fréquemment surexprimée dans plusieurs types de cellules cancéreuses, et on a pu associer Prx1 aux processus de carcinogenèse et de métastase, ainsi qu’à la résistance à la radiothérapie ou la chimiothérapie. Ainsi, Prx1 pourrait donc être une cible anticancéreuse intéressante. Au cours de ce travail de thèse, nous avons d’abord évalué l'impact d’une diminution de Prx1 (Prx1 knockdown (Prx1–)) sur la sensibilité cellulaire à des dizaines de médicaments anticancéreux dont la vinblastine, le taxol, la doxorubicine, la daunorubicine, l’actinomycine D, et le 5-fluorouracile, et d’agents connus pour provoquer la production d’espèces réactives de l’oxygène (ROS), dont le peroxyde d'hydrogène, le 2-phényléthyle isothiocyanate, le β-lapachone (β-lap) et la ménadione. Nous avons mis en évidence qu’une diminution de Prx1 augmente significativement la sensibilité des cellules à l'effet cytotoxique de la β-lap et de la ménadione, deux naphtoquinones possédant une activité anti-tumorale.Nous avons étudié les mécanismes responsables de l'augmentation de la cytotoxicité de la β-lap dans un contexte Prx1–. Nous montrons que la toxicité accrue de la β-lap dans des cellules Prx1– est due à une accumulation intracellulaire de ROS. Cet effet est dépendant de l’activité NADPH quinone oxydoréductase (NQO1) et s’accompagne d’une phosphorylation de c-Jun N-terminal kinases (JNK), protein 38 (p38), extracellular signal-regulated kinases (Erk) et des mitogen-activated protein kinases (MAPK), mais aussi d’une diminution des niveaux protéiques de la thiorédoxine 1. En se basant sur le fait que Prx1 est une enzyme antioxydante et un partenaire d'au moins ASK1 et JNK, deux éléments clés de la voie MAPK, nous proposons que la sensibilisation à la β-lap, observée après diminution de Prx1, est provoquée par une action synergique entre l'accumulation de ROS et l'induction de la voie MAPK, conduisant ainsi à l'apoptose.Nous avons ensuite étudié les mécanismes responsables de l'augmentation de la cytotoxicité de la ménadione dans le contexte Prx1–. La sensibilité accrue des cellules à l'effet cytotoxique de la ménadione et également associée à l'accumulation rapide et massive des ROS intracellulaire et à une mort cellulaire ressemblant à la nécrose programmée (necroptosis). L’accumulation de ROS induite par la ménadione et très rapidement détectée dans le cytosol, le noyau, et de façon encore plus importante, dans la matrice mitochondriale. Ce phénomène est en corrélation avec l'oxydation importante des thiorédoxine 2 et peroxiredoxine 3, deux protéines antioxydantes localisées dans la mitochondrie. La diminution de l’expression de Prx1 s’accompagne d’une augmentation des quantités tant de l’ARNm que de la protéine NRH: quinone oxydoréductase 2 (NQO2). Cette augmentation de l'activité de NQO2 est en grande partie responsable de l'accumulation intracellulaire de ROS et de la mort cellulaire après le traitement à la ménadione. Nos données révèlent que l’accumulation de ROS dans les cellules Prx1– provient de la résultante entre l’augmentation de leur production par NQO2 au cours du métabolisme de la ménadione et la diminution de leur élimination par Prx1. Enfin et de façon surprenante, selon la nature des naptoquinones (β-lap ou ménadione), les voies métaboliques qui conduisent à l'accumulation des ROS, ou les voies de signalisation et les mécanismes de mort cellulaire impliqués semblent être distincts. / Peroxiredoxins have multiple cellular functions as major antioxidants, signaling regulators, molecular chaperones and tumor suppressors. Peroxiredoxin 1 (Prx1) is the most abundant among the six isoforms of human peroxiredoxins. It is frequently over-expressed in various cancer cells, which is known associated with carcinogenesis, metastasis and resistance to radiotherapy or chemotherapy. Prx1 could thus be an interesting anticancer target. In this study, we first evaluated the impact of Prx1 knockdown (Prx1–) on cellular sensitivity to dozens of anticancer drugs including vinblastine, taxol, doxorubicin, daunorubicin, actinomycin D, and 5-fluorouracil, and of reactive oxygen species (ROS)-generating agents, including hydrogen peroxide, 2-phenylethyl isothiocyanate, β-lapachone (β-lap) and menadione. We observed that Prx1 knockdown significantly enhanced cancer cell sensitivity to β-lap and menadione, two naphthoquinones with anti-cancer activity.We first investigated the underlying mechanisms responsible for the specifically enhanced cytotoxicity to β-lap in a Prx1 knockdown context. Prx1 knockdown markedly potentiated β-lap-induced cytotoxicity through ROS accumulation. This effect was largely NAD(P)H:quinone oxidoreductase 1 (NQO1)-dependent and associated with the phosphorylation of c-Jun N-terminal kinases (JNK), protein 38 (p38) and extracellular signal-regulated kinases (Erk) proteins in mitogen-activated protein kinase (MAPK) pathways, and a decrease in thioredoxin 1 protein levels. Based on the fact that Prx1 is a major ROS scavenger and a partner of apoptosis signaling kinase 1 (ASK1) and JNK, two key components of MAPK pathways, we propose that Prx1 knockdown-induced sensitization to β-lap is achieved through the combined action of ROS accumulation and MAPK pathway activation, leading to cell apoptosis.We then investigated the underlying mechanisms responsible for the specifically enhanced cytotoxicity to menadione in Prx1– cells. Enhanced sensitivity to menadione was associated with a rapid and significant intracellular ROS accumulation and necroptotic-like cell death. Menadione-induced ROS accumulation occurred immediately in the cytosol, the nucleus, and even more noticeably in the mitochondrial matrix, correlated with significant oxidation of both mitochondria-localized thioredoxin 2 and peroxiredoxin 3. Prx1 knockdown significantly up-regulated mRNA and protein levels of NRH: quinone oxidoreductase 2 (NQO2). Increased activity of NQO2 was largely responsible for menadione-induced ROS accumulation and consequent cell death. Our data indicate that massive ROS accumulation results from the combined effect of increased ROS generation by higher NQO2 activity during menadione metabolism, and diminished Prx1 scavenging activity. Finally and noteworthy, the metabolic pathways that lead to ROS accumulation, downstream signaling pathways and cell death mechanisms appear to be distinct for β-lap and menadione.