Tesi sul tema "Anthracyclines"

L'interaction des anthracyclines avec des membranes est une étape importante dans la compréhension du mécanisme d'entrée et d'action de ces médicaments dans la cellule. Les anthracyclines sont des molécules amphiphiles qui peuvent interagir à la fois de manière électrostatique et de manière hydrophobe. Nous avons subdivisé notre étude en deux parties: la première en utilisant un modèle membranaire extrêmement simplifié, les liposomes, et la seconde en utilisant des membranes naturelles de cellules k562 sensibles et résistantes à la doxorubicine. L'étude de l'interaction des anthracyclines avec des liposomes a été réalisée par deux techniques différentes: I) par dichroïsme circulaire à faible rapport de phospholipide par anthracycline et à forte concentration d'anthracycline et II) par microspectrofluorimétrie à rapport plus élevé de phospholipide par anthracycline et a faible concentration d'anthracycline. L'interaction électrostatique intervient au niveau de la liaison des molécules chargées à la membrane, mais elle est masquée par l'interaction hydrophobe: elle est détectable dans le cas de certaines anthracyclines très peu lipophiles et à faible rapport de phospholipide par anthracycline. Les anthracyclines sont localisées dans les tètes polaires phospholipidiques plus ou moins profondément. Lorsque nous remplaçons les liposomes par des membranes naturelles, nous constatons un comportement différent. Il peut être du à un changement de fluidité attribuée à la présence d'autres composants membranaires, principalement des protéines, ou à des interactions avec des protéines

L'immunité antitumorale induite par les cellules dendritiques intratumorales contribue à l'efficacité de la chimiothérapie à base d'anthracycline dans le cancer. Nous avons identifié un allèle de perte de fonction du gène codant pour le récepteur formyl peptide 1 (FPR1) qui était associé à une faible survie sans métastases et à une survie globale chez les patientes atteintes d'un cancer du sein et colorectal recevant une chimiothérapie adjuvante.Les effets thérapeutiques des anthracyclines ont été abrogés chez les souris Fpr1 (-/-) porteuses de tumeurs en raison d'une immunité antitumorale altérée. Les cellules dendritiques déficientes en Fpr1 ne parvenaient pas à s'approcher des cellules cancéreuses mourantes et, en conséquence, ne pouvaient pas déclencher l'immunité des cellules T antitumorales.Des expériences réalisées dans un dispositif microfluidique ont confirmé que FPR1 et son ligand, l'annexine-1, favorisaient des interactions stables entre les cellules cancéreuses mourantes et les leucocytes humains ou murins.Nous avons également étudié la contribution possible de FPR1 à l'efficacité d'une combinaison de mitoxantrone (MTX) et cyclophosphamide (CTX) pour le traitement du cancer du sein induit par l'hormone.Le cancer du sein induit par une combinaison d'acétate de médroxyprogestérone (MPA) et de 7,12-diméthylbenz [a] anthracène (DMBA) a pu être traité avec succès avec MTX plus CTX dans la mesure où la croissance tumorale était retardée et la survie globale augmentée (par rapport à commandes traitées uniquement avec un véhicule).Toutefois, l'efficacité thérapeutique de la thérapie combinée a été complètement abolie lorsque les récepteurs FPR1 ont été bloqués au moyen de la cyclosporine H (CSH). Des études génétiques futures sur les cancers du sein traités par chimiothérapie néoadjuvante sont nécessaires pour valider ces résultats au niveau clinique.L'ensemble de ces résultats mettent en évidence l'importance de FPR1 dans les réponses immunitaires anti-cancéreux induites par la chimiothérapie
Antitumor immunity driven by intratumoral dendritic cells contributes to the efficacy of anthracycline-based chemotherapy in cancer. We identified a loss-of-function allele of the gene coding for formyl peptide receptor 1 (FPR1) that was associated with poor metastasis-free and overall survival in breast and colorectal cancer patients receiving adjuvant chemotherapy.The therapeutic effects of anthracyclines were abrogated in tumor-bearing Fpr1(-/-) mice due to impaired antitumor immunity. Fpr1-deficient dendritic cells failed to approach dying cancer cells and, as a result, could not elicit antitumor T cell immunity.Experiments performed in a microfluidic device confirmed that FPR1 and its ligand, annexin-1, promoted stable interactions between dying cancer cells and human or murine leukocytes.We investigated also the possible contribution of FPR1 to the efficacy of a combination of mitoxantrone (MTX) and cyclophosphamide (CTX) for the treatment of hormone-induced breast cancer.Breast cancer induced by a combination of medroxyprogesterone acetate (MPA) and 7,12-Dimethylbenz[a]anthracene (DMBA) could be successfully treated with MTX plus CTX in thus far that tumor growth was retarded and overall survival was extended (as compared to vehicle-only treated controls).However, the therapeutic efficacy of the combination therapy was completely abolished when FPR1 receptors were blocked by means of cyclosporin H (CsH). Future genetic studies on neoadjuvant chemotherapy-treated breast cancers are warranted to validate these findings at the clinical level.Altogether, these results highlight the importance of FPR1 in chemotherapy-induced anticancer immune responses

Anthracyclines are a group of chemotherapeutics that include adriamycin (doxorubicin), daunorubicin, idarubicin, and epirubicin. Anthracyclines are active against a wide range of tumours, in particular, adriamycin is used in the treatment of breast cancer, Hodgkin’s lymphoma, lung cancer, multiple myeloma and re-occurring ovarian cancer. Despite the broad spectrum of actions, resistance or severe cardio-toxicity limits the use of these important anticancer-drugs. The search for a “better anthracycline” has resulted in more than 2000 analogs, but only a few more anthracyclines have attained clinical approval. Although the exact mechanism by which adriamycin exerts its anti-tumour activity is uncertain, the dominant mechanism appears to involve impairment of topoisomerase IIα activity consistent with observed DNA intercalation and nuclear localization. The search for less toxic and more effective anthracyclines has led to the discovery of nemorubicin, a doxorubicin derivative in which the amino nitrogen of the daunosamine unit is incorporated into a methoxymorpholinyl ring. Preclinical investigations showed that nemorubicin, unlike classic anthracyclines, is not cardiotoxic and retains antitumour activity in various multidrug-resistant tumor models. Encouraging results have been obtained in phase I/II clinical trials in which the drug was administered by the intra-hepatic artery route. Nemorubicin is 80-120 times more potent than doxorubicin in vivo; in contrast, its in-vitro activity is only eight times greater than doxorubicin’s toward cultured drug sensitive tumour cells. A recent study established that nemorubicin is converted by enzyme CYP3A in a more cytotoxic metabolite PNU-159682, which was found to be 700-2400 times more potent that its parent drug toward cultured human cancer cells and which exhibits significant efficacy in in vivo tumor models. Ongoing studies aimed at exploring the molecular mechanism of action of PNU-159682 indicate that it has different effects on cell cycle progression and different DNA interacting properties, compared to both MMDX and doxorubicin. Moreover, further recent data suggest that PNU-159682 retains its activity against tumor cell lines with mechanisms of resistance different from those classical anticancer agents including MDR-1 gene overexpression, reduced topoisomerase II activity, and mutations in the topoisomerase I gene, these latter genetic alterations conferring resistance in vitro to the parent drug, MMDX [1]. We used different experimental approaches aimed to rationalizing the high activity of this metabolite. Test in vitro performed in our laboratory with kinetoplast DNA confirmed the inactivity of the metabolite against topoisomerase IIα. The absence of activity toward topoisomerase suggests that the high cytotoxicity of this compound had to be searched elsewhere. Anthracyclines such as doxorubicin and daunorubicin can bind covalently the DNA when activated with formaldehyde. Moreover, anthracyclines that have intrinsic ability to form cross-links to the DNA, such as cyanomorpholinyl-doxorubicin or barminomycin, were found to exhibit high cytotoxicity comparable with the PNU. Then we considered the possibility that PNU interacts with the DNA as a preactivated anthracycline. Our work evidenced that PNU behaves similarly to the activated doxorubicin (doxorubicin mixed with H2CO) in DNA melting analyses. PNU quickly reacts with double-strand oligonucleotides to form adducts detectable by DPAGE. These adducts are sufficiently stable to be isolated by HPLC. Mass characterization confirmed that these complexes are formed by duplex DNA bound to the anthracycline. These investigations suggest that the reaction between PNU and DNA does not involve the formation of a classical cross-link, but in relation to the electrophoretic, chromatographic and mass spectrometry results these adducts can be ascribed to the family of “virtual cross-link” (VXL). Anthracyclines-formaldehyde conjugate or anthracyclines in formaldehyde buffer have the specific ability to intercalate into DNA, forming covalent bonding; a methylene bridge links the amino group of the anthracycline to the 2-amino group of a G-base in the minor groove, while the other strand of DNA is stabilized by hydrogen bonds. Such unusual combination of intercalation, covalent bonding and hydrogen bonding is referred to as the virtual cross-linkink [2], that leads to the formation of more stable complexes between the anthracyclines and the DNA, improving the drugs’ cell killing ability. Among the different mechanisms of anticancer activity of anthracyclines, anthracycline-DNA adducts formation elicited interest related to the possibility to find safer and more efficacious anticancer drugs. Anthracycline-formaldehyde conjugates and cross-linking anthracyclines exhibit high cytotoxicity comparable to classical cross-linking drugs. We used different anthracyclines aimed to rationalize the structure activity relationship for the formation of VXL. We confirmed by electrophoretic and chromatographic analyses that aminosugar and its amino nitrogen is absolutely necessary for the formation of “VXL” and we discussed the role of the 4' position of the daunosamine in modulation of this activity. The presence of methylene bridge and its relationship with guanine was confirmed by mass spectrometry.
Le antracicline sono un’importante famiglia di chemioterapici, tra queste sono usate prevalentemente l’adriamicina (doxorubicina), la daunorubicina, l’idarubicina e l’epirubicina. Sono farmaci con un ampio spettro d’azione, in particolare l’adriamicina è usata nel trattamento del cancro al seno, del linfoma di Hodgkin, del cancro al polmone, del mieloma multiplo e del cancro ovarico recidivo. Nonostante l’ampio spettro d’azione la resistenza e la severa cardiotossicità limitano l'uso di questi importanti farmaci anticancro. La ricerca di migliori derivati ha dato luogo a più di 2000 analoghi dei quali solamente pochi di essi hanno raggiunto l’approvazione clinica. Anche se il meccanismo esatto con il quale l’adriamicina esercita l’attività anticancro è incerta, il meccanismo principale coinvolge un danno nei confronti dell’enzima topoisomerasi II, un meccanismo supportato dall’intercalazione nel DNA e dalla localizzazione nucleare. La ricerca di antracicline meno tossiche e più efficaci ha portato alla scoperta della nemorubicina, un derivato della doxorubicina in cui l'azoto amminico della daunosamina è incorporato in un anello metossimorfolinico. Le indagini precliniche hanno mostrato che la nemorubicina, diversamente dalle antracicline classiche non è cardiotossica e l'attività antitumorale è mantenuta nei vari modelli di tumore resistenti alla terapia. Risultati incoraggianti sono stati ottenuti in fase I/II dove il composto è stato somministrato attraverso l’arteria intraepatica. La nemorubicina è 80-120 più potente della doxorubicina in vivo, diversamente la sua attività in vitro è solamente otto volte rispetto alla doxorubicina verso culture cellulari tumorali sensibili alle antracicline. Un recente studio ha stabilito che la nemorubicina è convertita dall’enzima CYP3A in un metabolita estremamente più citotossico, il PNU-159682. Questo metabolita è risultato dalle 700 alle 2400 volte più potente rispetto al progenitore nemorubicina verso cellule cancerose umane in coltura e ha mostrato un’efficacia significativa in diversi modelli di tumore in vivo. Studi in corso finalizzati a definire il meccanismo molecolare di azione di PNU-159682 indicano differenti effetti sul ciclo cellulare e una differente interazione col DNA rispetto al progenitore nemorubicina e alla doxorubicina. Inoltre, dati recenti indicano che PNU-159682 mantiene la sua attività anche verso cellule aventi diversi meccanismi di resistenza rispetto a diversi agenti anticancro classici, inclusa la sovraespressione del gene MDR-1, la riduzione dell’attività di topoisomerasi II e mutazioni nel gene codificante per la topoisomerasi I: quest’ultima modifica genetica conferisce resistenza in vitro alla nemorubicina [1]. Noi abbiamo usato diversi approcci sperimentali per razionalizzare l’elevata attività di questo metabolita. Test condotti in vitro nel nostro laboratorio con kinetoplast DNA hanno confermato l'inattività del metabolita nei confronti della topoisomerasi II. L'assenza dell'attività verso la topoisomerasi ci suggerisce che l’alta citotossicità di questo metabolita è da ricercarsi altrove. Antracicline come doxorubicina e daunorubicina possono legare covalentemente il DNA quando attivate con formaldeide. Inoltre è stato trovato che antracicline che hanno un’intrinseca attività a formare cross-link col DNA come la cianomorfolino-doxorubicina o la barminomicina posseggono un’alta citotossicità comparabile col PNU. Quindi abbiamo considerato la possibilità che il PNU interagisca col DNA come una antraciclina preattivata. Il nostro lavoro ha evidenziato che il PNU si comporta in modo analogo alla doxorubicina attivata (doxorubicina con formaldeide) nelle analisi di melting del DNA. PNU reagisce velocemente con oligonucleotidi a doppio filamento per formare addotti visualizzati in DPAGE. Questi addotti sono sufficientemente stabili per essere isolati tramite HPLC. La caratterizzazione ottenuta tramite spettrometria di massa ha confermato che questi complessi sono formati da DNA a doppio filamento legato all’antraciclina. Questi studi suggeriscono che la reazione tra PNU e DNA non coinvolge la formazione di un classico cross-link, ma in relazione ai risultati elettroforetici, cromatografici e di spettrometria di massa questi addotti possono essere annoverati nella famiglia dei “virtual cross-link” (VXL). I coniugati antracicline-formaldeide o le antracicline in tampone contenente formaldeide hanno la specifica abilità di intercalarsi nel DNA formando legami covalenti; un ponte etilenico lega l’ammino gruppo dell’antraciclina col 2-amino gruppo della base guaninica nel solco minore, mentre l’ altra catena del DNA è stabilizzata tramite legami idrogeno. Questa particolare combinazione di intercalazione, legame covalente e legame ad idrogeno è chiamata virtual cross-link (VXL) [2], che porta alla formazione di complessi più stabili tra le antracicline e il DNA aumentando la tossicità cellulare delle antracicline. Tra i diversi meccanismi anticancro delle antracicline, la formazione di addotti DNA-antracicline ha suscitato notevole interesse riferito alla possibilità di trovare nuovi farmaci anticancro più sicuri e più efficaci. I coniugati antraciclina-formaldeide e le antracicline cross-linkanti esibiscono un’elevata citotossicità comparabile con i classici agenti cross-linkanti. Abbiamo usato differenti antracicline con lo scopo di razionalizzare il rapporto struttura attività nella formazione del VXL. Abbiamo confermato attraverso l’analisi elettroforetica e cromatografica che l’amminozucchero e l’azoto amminico sono assolutamente necessari per la formazione del “VXL” e abbiamo discusso il ruolo della posizione 4' nella daunosamina nella modulazione di questa attività. La presenza del ponte metilenico e la sua relazione con la guanina è stata confermata mediante spettrometria di massa.

Au cours de ce travail, notre objectif a été d'étudier le rôle du microenvironnement sur les capacités anti-migratoires de la doxorubicine chez la lignée cellulaire humaine HT-1080. Dans ce but, nous avons utilisé deux modèles de culture cellulaire : l'un sur substrat 2D recouvert de protéines matricielles (collagène de type I ou fibronectine) et l'autre au sein d'une matrice 3D (gel de collagène) mimant un environnement in vivo. Les expériences témoins sur support plastique seul montrent que des doses subtoxiques de doxorubicine exercent un effet anti-migratoire marqué en désorganisant totalement les fibres de stress d'actine et en remaniant la distribution de la vinculine. Par contre, en présence de protéines matricielles, l'effet anti-migratoire du médicament se retrouve totalement inhibé. Cette protection serait due à une préservation des niveaux d'activation de la GTPase RhoA nécessaire à la formation des fibres de stress, et de la FAK impliquée dans la formation des plaques d'adhésion focale. Les études réalisées au sein d'une matrice 3D révèlent que ce type de microenvironnement peut jouer un rôle de barrière physique en retardant, à court terme, la biodistribution de la doxorubicine. Des incubations long terme, permettant de s'affranchir de l'effet barrière, montrent que l’effet protecteur vis-à-vis des capacités anti-migratoires du médicament est peu marqué et s'accompagne d'une non modification du niveau d'activation de FAK. En conclusion, le microenvironnement s'avère capable de protéger la cellule tumorale vis-à-vis de l'effet anti-migratoire d'un médicament. Cependant, cet effet est fortement dépendant des conditions de culture ; ce qui souligne l'extrême adaptabilité de la cellule envers le milieu extérieur. A l'image des travaux réalisés pour l'effet cytotoxique des médicaments, nos résultats montrent que le paramètre microenvironnement devrait être pris en compte dans l'étude des propriétés pharmacologiques des agents anti-tumoraux
The @aim of our study was to evaluate the role of the microenvironment on the anti-migratory abilities of doxorubicin on HT-1080 cell line. In this context, two models of cell culture have been used: one on a 2D-coated substrate with extracellular matrix proteins (type I collagen or fibronectin) and the other one in a 3D collagen matrix mimicking an in vivo environment. Control experiments on plastic showed that subtoxic doses of doxorubicin exhibit a significant anti-migratory effect by totally disorganizing actin stress fibers and by modifying vinculin distribution. However, the anti-migratory effect of the drug is totally inhibited in presence of matrix proteins. This protection could be due to the preservation of the activation states of RhoA GTPase, which are necessary for the formation of actin stress fibers, and of FAK, implicated in the formation of focal adhesions. The study carried out in a 3D matrix demonstrates that this type of microenvironment can act as a physical barrier by delaying the biodistribution of doxorubicin. Long-term incubations, avoiding the barrier effect, show that the protectory effect to doxorubicin anti-migratory effect is less important and is not followed by a modification of FAK activation. In conclusion, the microenvironment is able to protect the tumor cell from the anti-migratory effect of a drug. However, this effect is very dependent on culture conditions, which underlines the extreme adaptability of the cell to its environment. In comparison to previous work on the cytotoxic effect of drugs, our results demonstrate that the microenvironment should be taken into account in the study of pharmacological properties of anti-tumor drugs

Dans le but de synthétiser des composés bifonctionnels à propriétés antitumorales, nous avons étudié les intéractions de l'adriamycine avec les métalocènes dichlorures cp2mcl2 (m=zr, ti, v). ; l'intéraction de la streptonigrin avec AuCl4. En utilisant des techniques spectroscopiques telles que l'absorption uv-visible, fluorescence et dichroisme circulaire

Several strategies outlining approaches to the synthesis of the heteroanthracyclinones 4-demethoxyxanthodaunomycinone and 4-demethoxyisoxanthodaunomycinone (7,8,9,10-tetrahydrobenzo(b)-6,7,9,11-tetrahydroxy-9-acetylxanthen-12 and 5-one) are described.
The condensation of tetralin 2-acetyl-5,8-dimethoxy-1,2,3,4-tetrahydro-2-naphthol with o-methoxybenzoic acid was investigated and useful large-scale syntheses of important 1,4-dimethoxy-substituted xanthone intermediates were developed.
Diels-Alder cycloaddition reaction between a xanthone-derived o-quinodimethane intermediate and an olefin afforded a low yield of adduct. On the other hand, excellent yields of isolable but labile adducts were obtained in the cycloaddition reaction between xanthoquinone (and also thioxanthoquinone) and Danishefsky's dienes. The formation of linear vs internal adducts was rationalized on the grounds of resonance and FMO theory. Efforts to induce unactivated dienes to cycloadd using catalysts as well as annulation studies on model compounds using the novel reagent (E)-N-vinylpyrrolidine-(beta)-(2-lithio-1,3-dithian-2-yl) (as a synthon of the (alpha),(beta)-dianion of acetaldehyde) are discussed.
The synthesis of daunomycin and xanthodaunomycin analogs carrying a carbon substituent at position 7 were not accessible using the Diels-Alder cycloaddition reaction as diene 1-carbomethoxy-3-triethylsilyloxy-1,3-butadiene failed to react with either quinizarinquinone or xanthoquinone even at elevated temperatures.
The compound 4-hydroxy-1- 2- (2-hydroxyethyl)amino ethyl amino xanthone and the 4-methoxy derivative were prepared and found to be inactive in the in vivo P-388 mouse leukemia model system.

Dans ce travail, nous avons étudié les interactions de cations métalliques avec des molécules appartenant a deux classes d'antitumoraux: 1) les anthracyclines 2) les cyclopropylpyrrolindoles. Les anthracyclines présentent des spectres d'absorption et de dichroïsme circulaire qui sont extrêmement sensibles à un certain nombre de paramètres tels que la concentration et, par conséquent, l'état d'association de ces molécules, état de déprotonation, solvant etc. Les modifications spectrales observées lors de l'interaction de ces molécules avec différents agents tels qu’ADN, membrane, cations métalliques, etc. Ont donc été largement utilisées pour tenter d'identifier la nature des sites d'interactions. Cependant, dans la plupart des cas, seule la forte bande située dans le visible à 480 nm a été prise en considération. Les modifications des autres bandes, en particulier celles situées dans la région 270-400, ont été peu étudiées. Dans la première partie, notre but est de corréler les modifications spectrales, en particulier celles des spectres de dichroïsme circulaires, aux modifications de la structure des différentes anthracyclines, pour cela nous disposions d'un grand nombre de dérivés ainsi que de nombreux complexes métalliques de ces anthracyclines, synthétisés dans notre laboratoire. Nous avons étudié l'influence de l'état de déprotonation de ces anthracyclines, de leur interaction avec l'ADN et de leur coordination à des métaux sur les spectres de dichroïsme circulaire. Dans une deuxième partie nous nous sommes intéressés à de nouvelles molécules à propriétés antitumorales développées par le laboratoire Upjohn, qui sont en phase préclinique. Nous avons focalise notre attention sur deux molécules de la famille de cyclopropylpyrrolindoles, la carzelesine et l'adozelesine. Nous avons étudié les spectres d'absorption et de dichroïsme cellulaire de ces composes en fonction du ph, de la température, de l'interaction avec l'ADN et avec différents ions métalliques, tels que Ca(II), mg(II), Zn(II), Fe(II), Fe(III), Cu(II). En particulier nous avons observe que la carzelesine subit une transformation, catalysée par les ions Cu(II), conduisant a une nouvelle espèce qui pourrait jouer un rôle important dans l'activité de ce médicament

La synthèse de la (+)-désacétyl-9 hydroxyméthil-9 déméthoxy-4 daunomycinone a été réalisée en sept étapes avec un rendement de 20 à 30%. La stratégie de construction du système tétracyclique fait intervenir deux condensations aldoliques successives (inter puis intramoléculaire) selon les conditions de Marschalk ou de Lewis entre la leucoquinizarine (noyaux DCB) et un aldéhyde dérivé de l’α-D-isosaccharino-1,4 lactone (précurseur du cycle A). L’utilisation de cet hydrate de carbone permet d’éviter l’introduction délicate des alcools en C-7 et/ou en C-9 en fin de synthèse. Le contrôle de la stéréochimie au cours de la séquence réactionnelle ainsi que la stéréospécificité de la cyclisation conduisant exclusivement à la configuration naturelle cis 7(s), 9(s) des anthracyclinones, constituent deux aspects intéressants de cette synthèse. Le produit de couplage de cet aglycone avec le chlorure du désoxy-2-L-fucose semble très prometteur dans la mesure où les premiers tests de cytotoxicité in vitro ont révélé une activité proche de celle de la doxorubicine
A seven steps synthesis of (+)-4-déméthoxy-9-déacétyl-9-hydroxyméthyl daunomycinone is described with an overall yield of ~20 to 30%. The strategy of the tetracyclic ring construction required two successive aldol condensation reactions (inter and intramolecular) under Lewis or Marschalk conditions between leucoquinizarine (DCB ring) and an aldehyde derived from the α-D-isosaccharino-1,4 lactone (precursor of ring A). The use of this highly functionalized carbohydrate avoids the incorporation of the hydroxyl groups at C-7 and/or C-9 at the last stage. Two noteworthy aspects of the synthesis were the stereocontrolled sequence and the stereospecific cyclization leading exclusively to the natural configuration 7(s), 9(s) cis of anthracyclinones. The anthracycline resulting from the coupling with 2-déoxy-L-fucose chloride is great interest since the first cytotoxicity tests show a similar activity in vitro to that of doxorubicin

Multidrug resistance (MDR) commonly occurs during the treatment of cancer. Current research has focused mostly on the role of drug transporters, as the main mechanism of MDR; however, few have demonstrated a definite link between the expression or function of drug transporters and MDR in cancer patients. Anthracyclines such as doxorubicin and epirubicin, autofluoresce and can be monitored by confocal microscopy. Two of the four resistant cell fines generated in our lab: the MCF-7EPI cells and to some extent MCF-7 DOX cells, exhibit a localization defect, whereby epirubicin is localized primarily in the cytoplasm rather than the nucleus. This drug localization defect temporally correlated with the onset of drug-resistance during selection for drug resistance in these cell lines. Consistent with the possible sequestration of drugs into acidic vesicles, acridine orange staining has revealed the presence of aggregates of acidified vesicles in the perinuclear region of MCF-7EPI cells. However, co-localization experiments using a number of intracellular organelle markers determined that epirubicin was localized to lysosomes and not consistently to acidic vesicles. An inhibitor of vacuolar H+ ATPase, was unable to restore the localization of epirubicin to the nucleus. Immunofluorescence using an ABCB1 antibody revealed the localization of ABCB1 predominantly in the plasma membrane and to some extent in the perinuclear region of MCF-7EPI cells. Nevertheless, inhibitors of this transporter failed to restore localization of epirubicin to the nucleus. Taken together, these findings strongly suggest that the acquisition of epirubicin resistance in breast tumour cells may involve the P-glycoprotein independent sequestration of drug into lysosomes. These lysosomes need not be acidic, nor does the removal of acid vesicles by inhibition of vacuolar H+ ATPases block the sequestration of drug into lysosomes.

Deux domaines de produits naturels sont étudiés: les et les aminoglycosides. La première partie traite des anthracyclines. Afin de effets secondaires de ces dérivés, notamment une forte cardiotoxicité, carbohydrate est remplacé par différents cyclitols. Deux approches vers la synthèse de ces nouveaux ant1cancéreux potentiels ont été mises au point. L'une est basée sur la substitution du groupe en position 7 par un alcoolate. L'autre méthode met en jeu une réaction de DIELS-ALDER. Suivant la nature du diène, l'induction asymétrique varie lors de la réaction de cyclisation. Par cette méthode seule la synthèse sur des modèles simples a pu être réalisée avec de bons rendements, lorsqu’elle a été appliquée à des diènes substitués par un cyclohexane polyfonctionnalisé, les rendements obtenus n'ont pas été satisfaisants. La seconde partie étudie la formation d'un diamlno-1,4 cyclitol, précurseur chiral des fortimicines. La stratégie est la transformation d'un sucre en une cyclohexanone. Celle-ci, après une amination suivie d'une déprotection sélective du site de glycosilation, pourra donner accès aux fortimicines
Two types of antibiotics were studied in this thesis: anthracyclines and aminoglycosides. The first chapter deals with anthracyclines in which the synthesis of new classes of anthracyclines, hopefully with reduced cardiotoxicity, were attempted. Two approaches were explored:1- Substitution of the benzylic group at position 7 by different alcoholates,2- DIELS-ALDER cycloaddition of a dienophile (epoxytetrone) to a diene bearing chiral cyclohexane substituant. This leads essantially in one step ta the tetracyclic skeleton with a good asymetrie induction. The second chapter deals with the preparation of 1,4-diaminocyclitol which based on a Ferrier rearrangement. The second amine is introduced by oximation follow by reduction

Iron (Fe) is essential for cell growth and replication as many Fe-containing proteins catalyse key reactions involved in energy metabolism (cytochromes, mitochondrial aconitase and Fe-S proteins of the electron transport chain), respiration (hemoglobin and myoglobin) and DNA synthesis (ribonucleotide reductase). If not appropriately shielded, Fe could participate in one-electron transfer reactions that lead to the production of extremely toxic free radicals. The Fe storage protein, ferritin, is essential to protect cells against Fe-mediated oxidative stress by accommodating excess Fe into its protein shell (Xu et al., 2005). However, despite intensive research over the last few decades, many questions relating to intracellular Fe metabolism, e.g. Fe release from ferritin remain unanswered. Therefore, it is important to elucidate the molecular mechanisms of Fe trafficking in cells. At the beginning of my candidature, little was understood regarding the effect of anti-cancer agents, anthracyclines on the Fe-regulated genes, including transferrin receptor-1 (TfR1), N-myc downstream-regulated gene-1 (Ndrg1) and ferritin. Furthermore, the mechanisms of ferritin-Fe release and anthracycline-mediated ferritin-Fe accumulation are unclear. The work presented in Chapters 3 and 4 has addressed these issues. Apart from the studies examining the molecular interactions of anthracyclines with Fe, a mouse model with perturbed Fe metabolism was used and the marked alterations of protein expression in the heart of this knockout mouse model was discussed in Chapter 5. Chapter 3 Anthracyclines are effective anti-cancer agents. However, their use is limited by cardiotoxicity, an effect linked to their ability to chelate iron (Fe) and perturb Fe metabolism (Xu et al., 2005). These effects on Fe-trafficking remain poorly understood, but are important to decipher as treatment for anthracycline cardiotoxicity utilises the chelator, dexrazoxane. Incubation of cells with doxorubicin (DOX) up-regulated mRNA levels of the Fe-regulated genes, transferrin receptor-1 (TfR1) and N-myc downstream-regulated gene-1 (Ndrg1). This effect was mediated by Fe-depletion, as it was reversed by adding Fe and was prevented by saturating the anthracycline metal-binding site with Fe. However, DOX did not act like a typical chelator, as it did not induce cellular Fe mobilisation. In the presence of DOX and 59Fe-transferrin, Fe-trafficking studies demonstrated ferritin-59Fe accumulation and decreased cytosolic-59Fe incorporation. This could induce cytosolic Fe-deficiency and increase TfR1 and Ndrg1 mRNA. Up-regulation of TfR1 and Ndrg1 by DOX was independent of anthracycline-mediated radical generation and occurred via HIF-1α-independent mechanisms. Despite increased TfR1 and Ndrg1 mRNA after DOX treatment, this agent decreased TfR1 and Ndrg1 protein expression. Hence, the effects of DOX on Fe metabolism were complex due to its multiple effector mechanisms. Chapter 4 The Fe storage protein, ferritin, can accommodate up to 4500 atoms of Fe in its protein shell (Harrison and Arosio, 1996). However, the underlying mechanism of ferritin-Fe release remains unknown. Previous studies demonstrated that anti-cancer agents, anthracyclines, led to ferritin-59Fe accumulation (Kwok and Richardson, 2003). The increase in ferritin-59Fe was shown to be due to a decrease in the release of Fe from this protein. It could be speculated that DOX may impair the Fe release pathway by preventing the synthesis of essential ferritin partner proteins that induce Fe release. In this study, a native protein purification technique has been utilised to isolate ferritin-associated partners by combining ultra-centrifugation, anion-exchange chromatography, size exclusion chromatography and native gel electrophoresis. In addition to cells in culture (namely, SK-Mel-28 melanoma cells), liver taken from the mouse was used as a physiological in vivo model, as this organ is a major source of ferritin. Four potential partner proteins were identified along with ferritin, e.g. aldehyde dehydrogenase 1 family, member L1 (ALDH1L1). Future studies are required to clarify the relationship of these proteins with cellular Fe metabolism and ferritin-Fe release. Chapter 5 A frequent cause of death in Friedreich’s ataxia patients is cardiomyopathy, but the molecular alterations underlying this condition are unknown. We performed two dimensional electrophoresis to characterise the changes in protein expression of hearts using the muscle creatine kinase frataxin conditional knockout (KO) mouse. Pronounced changes in the protein expression profile were observed in 9-week-old KO mice with severe cardiomyopathy. In contrast, only a few proteins showed altered expression in asymptomatic 4-week-old KO mice. In hearts from frataxin KO mice, components of the iron-dependent complex-I and -II of the mitochondrial electron transport chain and enzymes involved in ATP homeostasis (creatine kinase, adenylate kinase) displayed decreased expression. Interestingly, the KO hearts exhibited increased expression of enzymes involved in the citric acid cycle, catabolism of branched-chain amino acids, ketone body utilisation and pyruvate decarboxylation. This constitutes evidence of metabolic compensation due to decreased expression of electron transport proteins. There was also pronounced up-regulation of proteins involved in stress protection, such as a variety of chaperones, as well as altered expression of proteins involved in cellular structure, motility and general metabolism. This is the first report of the molecular changes at the protein level which could be involved in the cardiomyopathy of the frataxin KO mouse.

Les Anthracyclines (ex : Doxorubicine (Dox)) fréquemment utilisées en chimiothérapie anticancéreuse peuvent conduire à une cardiotoxicité aboutissant à de l’insuffisance cardiaque et à une cardiomyopathie dilatée. Au niveau cellulaire, la Dox est connue pour générer un stress oxydant fort, s’intercaler directement entre les brins d’ADN, inhiber les Topoisomérase II (TopII) ou encore provoquer une détresse énergétique conduisant à la mort aussi bien des cellules tumorales que des cardiomyocytes. Néanmoins, les voies de signalisations/mécanismes moléculaires complets ne sont pas identifiés à ce jour. L’objectif de ce travail de thèse consiste donc à mieux comprendre les mécanismes de la cardiotoxicité de la Dox et à identifier de nouvelles cibles cellulaires cardio-protectrices limitant les effets cardiaques délétères de cette Anthracycline. Dans ce but, nous focalisons nos recherches sur le rôle de la protéine EPAC1, un facteur d’échange pour les petites protéines G directement activé par l’AMPc, dans la réponse des cellules cardiaques à la Dox. EPAC1 est une protéine centrale de la voie de signalisation AMPc dans le cardiomyocyte en réponse à une stimulation β-adrénergique. Or, plusieurs études ont récemment montré l’implication de certains acteurs de cette voie (Rac, RhoA) dans la cardiotoxicité induite par la Dox faisant d’EPAC1 une cible thérapeutique potentielle. Nous avons donc étudié in vitro (cultures primaires de cardiomyocytes de rat nouveau-nés (Dox 1µM)) et in vivo (souris sauvages ou invalidées pour EPAC1 (Dox, iv, 12mg/kg total)) les effets de la Dox sur l’expression et l’activité d’EPAC1 et sur les voies de signalisation qu’il régule. In vivo, les souris sauvages traitées à la Dox développent une cardiomyopathie dilatée associée à une altération de l’homéostasie calcique 15 semaines après traitement. In vitro, la Dox induit des modifications de l’expression/activité d’EPAC1 et de l’homéostasie calcique, la formation de complexes TopIIβ/ADN conduisant à des dommages à l’ADN, une dérégulation de la biogénèse et de l’activité de la chaîne respiratoire mitochondriale et finalement à l’apoptose des cardiomyocytes. L’inhibition pharmacologique (Ce3F4, Esi09) ou génétique d’EPAC1 réduit l’ensemble des dommages cellulaires in vitro et empêche le développement de la cardiomyopathie dilatée in vivo. De manière importante, nous montrons que contrairement à ce qui est observé dans les cellules cardiaques, l’inhibition d’EPAC1 augmente la toxicité de la Dox envers les cellules tumorales et en particulier envers les cellules MCF-7 issues de cancer mammaire métastatique, principale indication de la Dox. Nos résultats suggèrent donc que l’inhibition d’EPAC1 semble être une stratégie thérapeutique prometteuse dans la prévention de la cardiomyopathie induite par les traitements anticancéreux à base d’Anthracyclines
Doxorubicin (Dox) is an Anthracycline commonly used to treat many types of cancer; unfortunately this chemotherapeutic agent often induces side effects such as cardiotoxicity leading to cardiomyocyte death and dilated cardiomyopathy (DCM). This cardiotoxicity has been related to reactive oxygen species generation, DNA intercalation, topoisomerase II inhibition and bioenergetics alterations resulting in DNA damages and ultimately in cardiomyocyte death. Nevertheless, complete molecular mechanisms are not yet identified. Therefore, there is a need for new treatment options and strategies aiming at reducing Dox side effects in the heart. Among these mechanisms, EPAC1 (Exchange Protein directly Activated by cAMP) signaling could be worth investigating as EPAC1 indirectly activates small G proteins (Rac1 and Rho A), which are known to be involved in Dox-induced cardiotoxicity. Therefore, we have investigated the effect of Dox on EPAC1 signaling in both in vivo mice model (C57BL/6 vs EPAC1 KO mice, iv injections, 12mg/kg) and in vitro model (primary culture of neonatal rat cardiomyocytes (NRVM), Dox 1μM). In vivo, Dox-treated mice developed a DCM associated with Ca2+ homeostasis dysfunction. In vitro, Dox induced DNA damages and cell death associated with huge mitochondrial disorders, characterized by a decrease in mitochondrial biogenesis and respiratory chain activity. This cell death is associated with apoptotic features including mitochondrial membrane permeabilization, caspase activation, cell size reduction and relative plasma membrane integrity. We also observed that Dox led to a modification of the protein level and the activity of EPAC1 in the same manner to the cAMP level. By contrast, the inhibition of EPAC1, prevented DNA/TopIIβ complexes, decreased Dox-induced DNA damages, loss of mitochondrial membrane potential, apoptosis and finally cardiomyocyte death. Mitochondrial biogenesis and respiratory chain activity operated normally when EPAC1 was inhibited. These results were confirmed in vivo since Dox-induced cardiotoxicity was prevented in EPAC1 KO mice as evidenced by unaltered cardiac function (no DCM) at 15 weeks post-treatment. Interestingly, the protection conferred by EPAC1 inhibition was not transferred in human cancer cell lines treated by Dox. Inhibition of EPAC1 could thus be a valuable therapeutic strategy to limit Dox-induced cardiomyopathy during cancer chemotherapy

L'hypoxie des cellules tumorales limite l'efficacite des traitements qui peuvent etre ameliores par accroissement de l'oxygene disponible au niveau des tumeurs. Mais les modes d'action de l'hyperoxygenation au niveau cellulaire sont encore mal compris. Nous avons utilise la videomicrofluorimetrie et le triple marquage sur cellules vivantes pour mesurer l'adn nucleaire (hoechst 33342), l'etat energetique des mitochondries (rhodamine 123) et les proprietes de la membrane plasmique (rouge nil). Les parametres morphologiques et fonctionnels obtenus pour chaque cellule ont permis, par classification ascendante hierarchique et analyse factorielle discriminante, de definir, pour des cellules lymphoblastiques ccrf-cem, une population de reference. Nous avons determine sa distribution dans les phases du cycle cellulaire (g0-g1, s, g2+m et cellules polyploi͏̈des gn). Cette population a permis d'analyser les effets de l'adriamycine (adr) sur les ccrf-cem. A 200 ng/ml les cellules etaient bloquees en gn, alors qu'a 1000 ng/ml la denaturation de l'adn figeait les populations cellulaires. L'apoptose a pu etre mise en evidence. Nous avons compare l'adr avec l'idarubicine (ida), anthracycline reputee generer moins de radicaux libres. Les resultats obtenus sous diverses conditions d'oxygenation (20% a 95%) ne revelent que peu d'effets de l'oxygene et pas de difference entre adr et ida. L'augmentation de la pression partielle en oxygene ne semble pas affecter les modes d'action de ces substances. On peut emettre l'hypothese que le gain d'efficacite lie a une hyperoxygenation est deja obtenu pour des cellules en conditions normoxiques (20%) par rapport a l'etat hypoxique (~5%) des tumeurs
Hypoxia of tumor cells limits the effectiveness of treatments which can be improved by an increase of available oxygen at tumor level. However, modes of action of hyperoxygenation at cellular level are still poorly understood. We used videomicrofluorometry and triple labeling on living cells to measure nuclear dna (hoechst 33342), mitochondrias energetic state (rhodamine 123) and plasma membrane properties (nile red). The morphological and functional parameters obtained for each cell allowed us, using typology and discriminant factorial analysis, to define a reference population of ccrf-cem lymphoblatsic cells. We determined their distribution in cell cycle phases (g0-g1, s, g2+m and polyploid cells gn). This population enabled us to analyze the effects of adriamycin (adr) on ccrf-cem. At 200 ng. Ml-1 cells were stopped in gn phase, while at 1000 ng. Ml-1 dna denaturation froze cellular populations. Apoptosis was also highlighted. We compared adr with idarubicine (ida), an anthracycline which is kown to generate few free radicals. Results obtained under different oxygenation conditions (20 to 95%) revealed only a few effects of oxygen and no difference between adr and ida. The increase of partial pressure of oxygene does not seem to modify the modes of actions of these drugs. We can put forward the hypothesis that the gain of efficiency linked to hyperoxygenation is already obtained for cells under normoxic conditions (20%) compared to tumor hypoxia (~5%)

Le but de ce travail est de contribuer au developpement des connaissances sur les methodes analytiques des anthracyclines, sur le metabolisme in vitro, et sur la pharmacocinetique de l'epirubicine chez l'homme. Les cinq molecules etudiees, la daunorubicine, la doxorubicine, l'epirubicine, l'idarubicine et l'esorubicine sont d'abord presentees puis des etudes analytiques sur l'extraction de ces produits et sur leur degradation photolytique dans le plasma, l'urine et le milieu de culture sont exposees. La cytotoxicite des cinq anthracyclines sur des hepatocytes humains, de rat, et sur des cellules epitheliales de foie de rat en culture est rapportee, ainsi que leur metabolisme in vitro par ces cellules. Les hepatocytes d'homme et de rat en culture primaire metabolisent les cinq anthracyclines suivant les memes voies qu'in vivo. Les specificites d'espece du metabolisme (entre homme et rat) et de substrat (entre les cinq anthracyclines) sont retrouvees dans les cultures d'hepatocytes. Des etudes realisees avec des lignees d'hepatomes humains hep g2 et bc2 et de rat fao, montrent que celles-ci ont conserve la specificite d'espece et de substrat pour l'activite metabolique aldocetoreductase. Par contre, la glucurono-conjugaison de l'epirubicine n'est pas retrouvee. La pharmacocinetique de l'epirubicine presente des differences qualitatives et surtout quantitatives entre les patients ayant des fonctions hepatiques normales ou perturbees. La clairance est abaissee chez les patients alcooliques. La voie de la glucuronoconjugaison de l'epirubicine est effondree chez les patients insuffisants hepatocellulaires

Notre deuxième travail expérimental visait à élucider le rôle de la surcharge pondérale dans le développement de la cardiotoxicité des anthracyclines et du trastuzumab. Grâce à un modèle murin de surpoids modéré et de risque cardio-métabolique accru induits par programmation post-natale, nous avons mis en évidence le rôle potentiateur d’une surcharge pondérale sur le développement de la cardiotoxicité aux anthracyclines ; alors que la cardiotoxicité du trastuzumab ne semble pas être en revanche majorée par le surpoids. Nos travaux ont également permis de préciser les conditions dans lesquelles existent des potentialisations des effets lors de l’association doxorubicine et trastuzumab
Cancer treatment has advanced considerably in recent years, allowing a reduction in mortality. Longer life expectancy of patients has helped to highlight the delayed onset of cardiovascular toxicity induced by these chemotherapies. The pathophysiological mechanisms responsible for these cardiac dysfunctions are complex, entangled and remain partially unknown. A better understanding of the phenomena involved in these cardiotoxicities is needed to prevent their occurrence. Therefore, we have developed two different experimental approaches to understand the pathophysiological mechanisms involved in the cardiac toxicity of anthracyclines and trastuzumab.A first experimental study aimed to clarify the role of iron in heart failure induced by anthracyclines. We have demonstrated that a tissular iron overload in mice prior to doxorubicin injection does not increase the cardiotoxicity of chemotherapy. On the contrary, the involvement of anti-radical defenses following the iron load could reduce cardiac oxidative damage generated by doxorubicin. In view of these data, the role of iron chelators in cardioprotection against anthracyclines has to be questioned.Our second experimental work was to elucidate the role of overweight in the development of anthracycline and trastuzumab cardiotoxicity. Using a mouse model of moderate overweight and of increased risk of cardiometabolic induced postnatal programming, we have highlighted the role of overweight on the development of anthracycline cardiotoxicity; whereas trastuzumab cardiotoxicity did not appear to be increased by overweight. Our work also clarified the conditions in which there are cumulative cardiac alterations when doxorubicin and trastuzumab are associated

Background: The molecular and cellular mechanisms corresponding to the compensatory and maladaptive hypertrophy and remodeling of the left ventricle with chronic doxorubicin (DOX) treatment are currently unclear. Non-invasive methods of determining these changes are still deficient. To investigate these changes, 8 groups of rats in 4 different studies including a control saline group of the same age, gender and strain were evaluated for cardiac morphology and function including: (1) DOX dose response using a cumulative dose of 7.5mg/kg, and 15mg/kg in 8-10 week old female Sprague-Dawley (SD) rats, (2) strain differences were investigated in response to a cumulative dose of 15mg/kg in 8-10 week old female Fisher (F344) rats compared to the SD rats treated with same dose, (3) the role of gender and aging were studied in response to DOX at a cumulative dose of 3mg/kg in male and female neonates, and (4) combined losartan and a cumulative dose of 15mg/kg of DOX in 8-10 week old female SD rats compared to controls of saline and 15mg/kg treated SD rats. Method: Onset of cardiac toxicity was assessed by echocardiography and the rat model of heart failure was developed when the fractional shortening declined ≤ 40%. The mean arterial pressure and single-photon-emission computer tomography scanning and Tc-99m-HYNIC-Annexin V were performed at week 10 to analyze blood pressure and quantify apoptosis, respectively. All rats were euthanized at week 10 except for the neonates and two of the 7.5mg/kg-treated SD rats that were left alive for study of long -term cardiac side effects. The heart and kidney tissues were harvested for protein isolation and histopathological studies. Blood samples were collected for hematological and lipid profile analysis in all the rats. Results: A dose- and time-dependent increase in LVmass coincided with a parallel increase in MAP, kidney damage, expression of myocardial erbB2, heat shock protein 90 Akt, mTOR, GSK-3β, TGF-β, pSMAD2, and cardiomyocyte apoptosis in SD rats treated with 7.5mg/kg and 15mg/kg of DOX at week 10. The 7.5 kg/kg treatment showed adaptive hypertrophy whereas the 15mg/kg treatment group showed maladaptive hypertrophy. However decompensation was apparent by week 14 in other rats treated with 7.5mg/kg. LVmass, FS, MAP, kidney damage, red blood cells and blood lipid levels were not significantly altered in the F344 rats compared to the 15 mg/kg-treated SD rats. Losartan supplementation reduced the left ventricular hypertrophy, improved myocardial contractility, and reduced TGF-β expression compared to the DOX-treated SD rats. The 3mg/kg of DOX in neonates induced cardiac toxicity and deaths in about 60% of males 50 weeks after treatment; the females instead developed mammary tumors. Conclusion: The results of this study suggest that age, gender, and strain differences are risks factors for doxorubicin-induced harmful reno-cardiovascular toxicity. The inhibition of TGF-β expression by losartan can be used in prevention of chronic doxorubicin-induced cardiac toxicity without interfering with its anti-tumor activities.

L’hypoxie tumorale peut induire une résistance aux traitements par l’adriamycine (ADR), et l’effet anti-cancéreux de l’anthracycline peut augmenter sous oxygénation hyperbare. Cependant, les mécanismes d’action impliqués dans ce gain d’efficacité thérapeutique restent à élucider. Nous avons évalué l’implication de la production d’espèces réactives de l’oxygène (ROS) dans l’amélioration de l’effet de l’ADR sur des cellules lymphoblastiques humaines CCRF-CEM sous hypoxie (2% O2) et sous normoxie (21% O2). Nous avons utilisé une nouvelle méthode de détection de ROS basée sur la mesure de la durée de vie de fluorescence de l'acide 1-pyrène butyrique. L’analyse d’images numériques des populations cellulaires après triple-marquage a fourni des informations morphométriques (tailles cellulaire et nucléaire) et fonctionnelles (activité mitochondriale, teneur en ADN) permettant (i) de quantifier l’induction de l’apoptose, et (ii) d’établir la distribution du cycle cellulaire par analyse multiparamétrique des données. Nous avons observé que le blocage du cycle cellulaire par l'ADR ne dépend pas des conditions d'oxygénation, alors que l’induction de l’apoptose et la production de ROS dues au traitement sont plus importantes sous condition oxygénée (21% O2). Si on admet que la condition normoxique est une hyperoxygénation comparée à l’état d’hypoxie tumorale in vivo, alors le gain d’efficacité thérapeutique de l’ADR fournit par une oxygénation hyperbare pourrait résulter d'une plus forte production de ROS par l'anthracycline, qui entraînerait une induction des processus apoptotiques plus importante
Tumour hypoxia is causally related with resistance to adriamycin (ADR) treatment. However, how hyperbaric oxygen therapy leads to therapeutic gain of the drug is unclear. We investigated the relation of reactive oxygen species (ROS) generation with anti-tumoural effect of ADR on human lymphoblastic CCRF-CEM cells under hypoxic (2% O2) and normoxic (21% O2) conditions. A new method was used to measure intracellular ROS variations through the fluorescence lifetime of 1-pyrenebutyric acid. Numerical image analysis of cell populations labelled with different vital stains allowed to collect morphometric (cellular and nuclear sizes) and physiological (mitochondrial activity, DNA content) informations used to (i) quantify apoptosis induction, and (ii) determine the cell cycle distribution through multiparametric analysis of collected data. We observed that oxygen level has no effect on the cell cycle arrest induced by ADR, whereas apoptosis induction and ROS production resulting from treatment are higher under oxygenated conditions (i. E. Normoxia). Considering normoxia as a hyperoxygenated condition compared to in vivo hypoxic tumour level, we suggested that improvement of anti-cancerous effect of ADR due to hyperbaric oxygen therapy results from higher intracellular ROS generation by the drug, leading to a greater induction of apoptosis

Nous avons étudié les effets protecteurs Brain Natriuretic Peptide (BNP) (hormone synthétisée par les cardiomyocytes) sur la toxicité aiguë et chronique de la doxorubicine (largement utilisé dans le traitement des cancers). Ces études ont été réalisées chez l’animal, in vivo, sur différents types de muscles, sur des cellules musculaires en culture et enfin, sur des fragments d’auricules humain. Lors du traitement aigu, la doxorubicine altère la respiration mitochondriale des cardiomyocytes et des cellules musculaires squelettiques par augmentation du stress oxydant. L’injection du BNP protége en ouvrant les canaux mKATP, en augmentant l’expression des ARNm de la SOD2 (système antioxydant) et en participant au maintien du potentiel transmembranaire. Le traitement chronique par doxorubicine diminue les capacités oxydatives maximales des complexes I, II et III de la chaîne respiratoire et augmente le stress oxydant. Le pré-conditionnement par BNP protège les muscles cardiaque et squelettiques de cette altération
The purpose of this research was to study the protective effects of brain natriuretic peptide (BNP), a hormone synthesized by cardiomyocytes, on the acute and chronic toxicity of doxorubicine, which is widely used in cancer treatment. These studies were carried out on live animals on various types of muscle, on cultured muscular cells, and subsequently on fragments of human heart appendages. During acute treatments doxorubicine modifies the mitochondrial function of the cardiomyocytes and the muscular cell structure by an increase in oxidative stress. The injection of BNP provides protection by opening the mKATP channels, by increasing the mitochondrial respiratory chain complex activity , the antioxidant systems, and participle in the maintenance of mitochondrial membrane potential. Chronic treatment with doxorubicine reduces the oxydatives capacities of the I, II and III complexes activities of respiratory chain and increase oxidative stress. Pre-treatment by BNP protects both heart and skeletal muscles from this deterioration

La doxorubicine est une anthracycline utilisée largement dans le traitement des cancers du sein et des hémopathies malignes, mais présente une cardiotoxicité qui en limite l’utilisation. Cette toxicité est attribuée en grande partie aux conséquences des anthracyclines sur le métabolisme énergétique cardiaque : les anthracyclines produisent au niveau cellulaire des espèces radicalaires de l’oxygène à l’origine d’une inhibition de la chaîne respiratoire mitochondriale, d’une dissipation du potentiel de membrane mitochondriale, d’une surcharge calcique mitochondriale. L’hypothèse de notre travail est que l’inhibition de l’ouverture du pore de transition de perméabilité mitochondrial (mPTP) puisse diminuer les altérations du métabolisme cardiaque et la dysfonction contractile observées dans des modèles d’exposition aigue et sub-aigue à la doxorubicine. L’étude a été réalisée sur des préparations de myocarde murin et humain. Nous avons montré que la doxorubicine altère rapidement les fonctions contractile et métabolique de préparations myocardiques contractiles humaine et murine à des concentrations de l’ordre de celles observées chez le patient. Ces altérations pouvaient être en partie expliquées par une ouverture du pore de transition de perméabilité mitochondrial comme en témoigne la cardioprotection offerte par la ciclosporine A dans ces modèles d’exposition aiguë. Nous avons également montré dans des modèles sub-aigue et chronique de cardiotoxicité de la doxorubicine que l’administration de ciclosporine A (à l’inverse du FK506) améliore la survie, la fonction contractile cardiaque et l’homéostasie mitochondriale en terme de potentiel de membrane mitochondrial, respiration mitochondrial, capacité d’accumulation du calcium, équilibre entre fusion et fission itochondriales. Ainsi, l’ensemble de nos résultats conforte sur myocarde murin et humain les données de la littérature quant au rôle important du pore de transition mitochondrial et des altérations du métabolisme énergétique dans la physiopathologie de la cardiotoxicité des anthracyclines. De plus, la ciclosporine A apparaît capable de préserver la fonction contractile des coeurs exposés aux anthracyclines, ainsi que la capacité du réseau mitochondrial à produire de l’énergie. Nous pensons que ces travaux permettent d’envisager la mise en place d’une étude de cardioprotection par la ciclosporine A vis à vis de la toxicité de la doxorubicine dans un essai clinique de type « proof of concept ».