[ES] Las antraciclinas son fármacos antineoplásicos ampliamente utilizados en el trata-miento de varios tipos de cáncer, incluyendo tanto tumores sólidos como hematoló-gicos. A pesar de su eficacia, su uso se ve limitado por su efecto cardiotóxico. El aumento de los supervivientes de cáncer, especialmente pediátrico, ha provocado que cada vez haya más personas con cardiotoxicidad inducida por antraciclinas. Por este motivo, es necesaria la búsqueda de nuevos modelos de enfermedad relevan-tes para comprender la fisiopatología del daño cardíaco inducido por antraciclinas, así como el desarrollo de nuevas terapias cardioprotectoras que permitan el uso de las antraciclinas evitando su efecto cardiotóxico. En este trabajo se ha estudiado, por una parte, la susceptibilidad al daño por do-xorrubicina (una de las principales antraciclinas empleadas en clínica) de cardiomio-citos obtenidos a partir de células madre pluripotentes inducidas derivadas de pa-cientes pediátricos oncológicos que experimentaron cardiotoxicidad causada por an-traciclinas. Los cardiomiocitos de los pacientes fueron tratados con doxorrubicina y se evaluaron diferentes parámetros, incluyendo la viabilidad, apoptosis, estrés oxida-tivo, daño genómico, daño mitocondrial, desorganización sarcomérica, etc. compa-rándolos con cardiomiocitos control. Nuestros resultados mostraron que estos car-diomiocitos recapitulan la susceptibilidad a la doxorrubicina observada en los pacien-tes, constituyendo un buen modelo de enfermedad para estudiar los mecanismos de cardiotoxicidad de la doxorrubicina o el cribado de fármacos. Por otra parte, se ha evaluado el papel cardioprotector de un miARN, el miR-4732-3p, frente al daño inducido por antraciclinas. Este miARN está desregulado en pa-cientes con cáncer de mama que sufrieron cardiotoxicidad inducida por antraciclinas. Para comprobar su efecto cardioprotector, este miARN fue sobreexpresado en célu-las cardíacas de rata, las cuales fueron tratadas con doxorrubicina, observándose que incrementaba la supervivencia de las células y reducía el estrés oxidativo. Tam-bién se estudió la cardioprotección in vivo en un modelo de cardiotoxicidad inducida por doxorrubicina en rata, observando que mejora la función cardíaca, reduce la fi-brosis intersticial y el estrés oxidativo. Además, se hizo un estudio de los posibles genes diana de este miARN. En su conjunto, nuestros resultados muestran que el miR-4732-3p tiene un efecto cardioprotector frente al daño por doxorrubicina, y po-dría ser una herramienta terapéutica para el tratamiento del daño cardíaco causado por las antraciclinas. / [CAT] Les antraciclines són fàrmacs antineoplàstics àmpliament utilitzats en el tractament de diversos tipus de càncer, incloent tant tumors sòlids com hematològics. Tot i la seva eficàcia, el seu ús es veu limitat pel seu efecte cardiotòxic. L'augment dels su-pervivents de càncer, especialment pediàtric, ha provocat que cada cop hi hagi més persones amb cardiotoxicitat induïda per antraciclines. Per aquest motiu, cal cercar nous models de malaltia rellevants per comprendre la fisiopatologia del dany cardíac induït per antraciclines, així com el desenvolupament de noves teràpies cardiopro-tectores que permetin l'ús de les antraciclines evitant-ne l'efecte cardiotòxic. En aquest treball s'ha estudiat, d'una banda, la susceptibilitat al dany per doxorrubi-cina (una de les principals antraciclines emprades en clínica) de cardiomiòcits obtin-guts a partir de cèl¿lules mare pluripotents induïdes derivades de pacients pediàtrics oncològics que van experimentar cardiotoxicitat causada per antraciclines. Els cardi-omiòcits dels pacients van ser tractats amb doxorrubicina i es van avaluar diferents paràmetres, incloent-hi la viabilitat, apoptosi, estrès oxidatiu, dany genòmic, dany mitocondrial, desorganització sarcomèrica, etc. comparant-los amb cardiomiòcits control. Els nostres resultats van mostrar que aquests cardiomiòcits recapitulen la susceptibilitat a la doxorrubicina observada en els pacients, constituint un bon model de malaltia per estudiar els mecanismes de cardiotoxicitat de la doxorrubicina o el cribratge de fàrmacs. D'altra banda, s'ha avaluat el paper cardioprotector d'un miARN, el miR-4732-3p, davant del dany induït per antraciclines. Aquest miARN està desregulat en pacients amb càncer de mama que van patir cardiotoxicitat induïda per antraciclines. Per comprovar el seu efecte cardioprotector, aquest miARN va ser sobreexpressat en cèl·lules cardíaques de rata, les quals van ser tractades amb doxorrubicina, obser-vant-se que incrementava la supervivència de les cèl·lules i reduïa l'estrès oxidatiu. També es va estudiar la cardioprotecció in vivo en un model de cardiotoxicitat induï-da per doxorrubicina en rata, observant que millora la funció cardíaca, redueix la fi-brosi intersticial i l'estrès oxidatiu. A més, es va fer un estudi dels possibles gens di-ana d'aquest miARN. En conjunt, els nostres resultats mostren que el miR-4732-3p té un efecte cardioprotector davant el dany per doxorrubicina, i podria ser una eina terapèutica per al tractament del dany cardíac causat per les antraciclines. / [EN] Anthracyclines are drugs widely used in the treatment of several types of cancer, including both solid tumors and hematologic malignancies. Despite its proven effica-cy, its use is hampered by its cardiotoxic effect. The increase in cancer survivors, especially pediatric, has led to more and more people with anthracycline-induced cardiotoxicity. Therefore, it is necessary to search for new relevant disease models to better understand the physiopathology of cardiac damage-induced by anthracy-clines, as well as the development of new cardioprotective therapies that allow the clinic use of anthracyclines avoiding their cardiotoxic effect. In this work we have studied, on the one hand, the susceptibility against doxorubicin damage (major anthracycline used in clinic) of cardiomyocytes obtained from in-duced pluripotent stem cells derived from oncology pediatric patients that underwent cardiotoxicity-induced by anthracyclines. Cardiomyocytes from these patients were treated with doxorubicin, and we evaluated several parameters, including cell viabil-ity, apoptosis, oxidative stress, genomic damage, mitochondrial damage, sarcomere disorganization, etc. comparing the results with control cardiomyocytes. Our results showed that these cardiomyocytes recapitulate the susceptibility against doxorubicin observed in the patients, making them a good disease model to study cardiotoxicity mechanisms of doxorubicin or drug screening. On the other hand, we evaluated the cardioprotective role of one miRNA, miR-4732-3p, against doxorubicin-induced damage. This miRNA is dysregulated in breast can-cer patients that suffered cardiotoxicity-induced by anthracyclines. To test its cardio-protective effect, this miRNA was overexpressed in rat cardiac cells that were treated with doxorubicin, showing an increment of cell survival and a reduction of oxidative stress levels. We also studied in vivo cardioprotection in a doxorubicin-induced cardi-otoxicity model in rat, showing an improvement in cardiac function, reduced intersti-tial fibrosis and reduced oxidative stress levels. Moreover, we studied possible target genes of this miARN. Overall, our results showed that miR-4732-3p has a cardiopro-tective role against doxorrubicin-induced damage and could be used as a therapeutic tool for treatment of cardiac damage caused by anthracycline. / Reinal Ferré, I. (2023). Modelado de la cardiotoxicidad inducida por antraciclinas en hiPSC-CM de pacientes oncológicos pediátricos y estudio del papel cardioprotector del miR-4732-3p [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/193075

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Introduction: The anthracyclines (ACs), daunorubicin (DNR) and doxorubicin (DXR) are two of the most effective drugs known for the treatment of systemic neoplasms and solid tumours. However, their clinical use is often hampered by their dosedependent cumulative cardiotoxicity, which leads to irreversible and fatal druginduced congestive heart failure. The mechanism by which ACs induces heart damage is not fully understood. Recent reports have indicated that DXR activates autophagy and ubiquitin proteasome-mediated degradation of specific transcription factors, however, no reports exists on the effect of ACs on the E3 ubiquitin ligases, MuRF-1 and MAFbx. The aim of the first part of the study was therefore to investigate the effect of DNR treatment on the protein and organelle degradation systems in the heart and to elucidate the signalling mechanisms involved. Although this model was ideal in allowing the investigation of the signalling pathways which are affected by DNR, it did not allow for further exploration or manipulation of signalling pathways that may be of potential benefit in this context. The in vitro model was therefore used to validate the hypothesis that increased autophagy alleviates AC-induced cardiotoxicity and delays the onset of cardiomyocyte death. The aims for the second part of the study were (i) to characterize the effect of DXR in H9C2 cells, (ii) to determine whether the induction/inhibition of autophagy in combination with DXR alleviates cytotoxicity and (iii) to investigate the influence of increased/decreased autophagy in combination with DXR on reactive oxygen species (ROS) production, mitochondrial function, endoplasmic reticulum (ER) stress and the ubiquitin proteasome pathway. In the final part of this study, an in vivo model was used to assess the potential benefit of autophagy in a novel GFP-LC-3 tumour bearing mouse model of acute DXR-induced cardiotoxicity. Material and Methods: Adult rats were divided into two groups where one group received six intraperitoneal injections of 2 mg/kg DNR on alternate days and the other group received saline injections as control. Hearts were excised and perfused on a working heart system the day after the last injection and freeze clamped for biochemical analysis. H9C2s were cultured and treated with Bafilomycin A1 (10 nM, inhibitor of autophagy) for 6 hrs, Rapamycin (50 μM, inducer of autophagy) for 24 hrs, DXR (3 μM) for 24 hrs or a combination of these drugs. Following treatment, cells were harvested and assessed for cell death, proteolytic activity and oxidative stress using western blotting, fluorescence microscopy and flow cytometry. In the final phase of the study, twenty-four female mice were injected at 8 weeks with a mouse breast cancer cell line (EO771) and after observation of tumour growth, animals were either treated with one injection (i.p.) of Rapamycin (4 mg/kg), two injections (i.p.) of DXR (10 mg/kg) or a combination of the two drugs. After the experimental protocol, mice were terminated and their hearts were rapidly excised. The hearts were divided cross-sectionally and utilized for biochemical and histological analyses. Results and Discussion: DNR treatment significantly attenuated myocardial function and increased apoptosis in the ex vivo heart model. DNR-induced cardiac cytotoxicity was associated with the upregulation of two E3 ubiquitin ligases, MuRF-1 and MAFbx as well as a significant increase in two markers of autophagy, beclin-1 and LC-3. These changes observed in the heart were also associated with attenuation of the PI3-kinase/Akt signalling pathway. The augmentation of autophagy with rapamycin before DXR treatment significantly reduced cell death in the in vitro model. Indeed, rapamycin treatment demonstrated to be a vital survival mechanism for acute DXR-induced cardiotoxicity as it decreased cellular ROS production, improved mitochondrial function and prevented nuclear translocation of DXR. Moreover, these changes in cardiomyocytes were also associated with a reduction in the ubiquitin-proteasome pathway (UPP). In the final part of this study, a novel tumour bearing GFP-LC3 mouse model was developed to confirm the results obtained in the in vitro study. It was demonstrated that acute DXR-induced cardiotoxicity resulted in increased apoptosis, the inhibition of autophagy and increased proteolysis via the UPP. These findings were associated with a reduction in body weight and cardiomyocyte cross-sectional area. The cardiotoxic effects of DXR were substantially reduced when autophagy was induced with rapamycin. Taken together, our data strongly indicates that it is possible to attenuate the cardiotoxic effects of doxorubicin in cancer patients by carefully controlling the levels of autophagy using rapamycin as adjuvant therapy. / AFRIKAANSE OPSOMMING: Inleiding: Die antrasikliene (AC’s), daunorubisien (DNR) en doksorubisien (DKS), is twee van die mees effektiewe AC wat bekend is vir die behandeling van sistemiese neoplasmas en soliede tumore. Hulle kliniese gebruik word egter deur dosis afhanklike kumulatiewe kardiotoksisiteit benadeel, wat tot onomkeerbare en dodelike kongestiewe hartversaking kan lei. Die meganisme waardeur AC’s hartversaking kan veroorsaak, word nog nie ten volle verstaan nie. Onlangse navorsing het aangetoon dat DKS autofagie en die ubikwitienproteosoom-bemiddelde degradasie van spesifieke transkripsie faktore aktiveer. Daar is egter geen literatuur wat die effek van AC’s op die E3-ubikwitienligases, MuRF-1 en MAFbx beskryfnie. Die doel van hierdie eerste afdeling van die studie is om die effek van DNR behandeling op die proteïen- en organel degradasie sisteme in die hart te ondersoek en om van die betrokke seinmeganismes te bepaal. Alhoewel hierdie model ideaal is om sommige seinweë wat deur DNR geaffekteer word, te ondersoek, kon seinoordragpaaie wat potensieël voordelig in hierdie konteks is, nie in bg. model gemanipuleer word nie. Die in vitro model is gebruik om die hipotese dat verhoogde outofagie AC-geïnduseerde kardiotoksisiteit verlaag en sodoende seldood verminder, te bevestig. Die doel van hierdie afdeling van die studie was: (i) om die effek van DKS op H9C2 selle te karakteriseer, (ii) om te bepaal of die induksie/inhibisie van outofagie in kombinasie met DKS kardiotoksisiteit verbeter (iii) om die invloed van verhoogde/verlaagde outofagie in kombinasie met DKS op reaktiwe suurstof species (ROS), mitokondriale funksie, endoplasmiese retikulum (ER) stress en die ubikwitienproteosoompad te ondersoek. In die finale deel van hierdie studie, is ‘n in vivo model gebruik om die moontlike voordelige effek van verhoogde outofagie in ‘n GFP-LC-3 tumor-draende muismodel met akute DKSgeïnduseerde kardiotoksisiteit, ondersoek. Materiaal en Metodes: Volwasse rotte is in twee groepe verdeel waar een groep ses intraperitoneale inspuitings van 2 mg/kg DNR op afwissellende dae ontvang het en die andergroep as ‘n kontrole, ‘n soutoplossing gekry het. Die harte is verwyder en geperfuseer op ‘n werkende hartsisteem een dag na die laaste inspuiting en gevriesklamp vir biochemiese analises. H9C2 selle is vir 6 uurgekweek en behandel met Bafilomisien A1 (10 nM, ‘n autofagie inhibitor), 24 uur met Rapamisien (50 μM, ‘n autofagie induseerder), 24 uur met DKS (3 μM) of ‘n kombinasie van hierdie middels. Na behandeling is selle ge-oes vir analises in seldood, proteolitiese aktiwiteit en oksidatiewe stress deur van westelike kladtegniek, fluoresensie mikroskopie en vloeisitometrie gebruik te maak. In die finale fase van hierdie studie is vier en twintig, agt weke oue wyfie muise ingespuit met ‘n muisborskankersellyn (E0771) en is tumorgroei waargeneem; die diere is of behandel met een rapamisien inspuiting (i.p) (4 mg/kg), of twee DKS inspuitings (i.p.) (10 mg/kg) of ‘n kombinasie van die twee middels. Na die eksperimentele protokol, is die muise van kant gemaak en hulle harte vinnig verwyder. Die harte is in twee verdeel en gebruik vir biochemiese- en histologiese analises. Resultate en Bespreking: DNR behandeling het kardiale funksie betekenisvol verswak en apoptose in die hart verhoog. DNR-geïnduseerde kardiotoksisiteit is geassosieer met die opregulering van E3-ligases, MuRF-1 en MAFbx en het ook ‘n betekenisvolle toename in twee outofagie merkers, beclin-1 en LC-3 veroorsaak. Hierdie veranderinge wat in die hart waargeneem is, is ook geassosieer met ‘n onderdrukking van die PI3-kinase/Akt seinweg. Die toename in outofagie met rapamisien voor DKS behandeling het seldood in die vorm van apoptose betekenisvol verlaag. Daarmee saam het verhoogde outofagie ‘n noodsaaklike oorlewings meganisme vir akute DKS-geïnduseerde kardiotoksisiteit gedemonstreer. Die rede hiervoor is dat dit ROS produksie verlaag het, mitokondriale funksie verbeter het en DKS translokasie vanuit die sitoplasma tot binne die nukleus verhoed het. Hierdie veranderinge in kardiomiosiete is ook met ‘n afname in die ubikwitienproteosoomseinweg (EPS) geassosieer. In die finale deel van hierdie studie, is ‘n nuwe tumor-draende muismodel ontwikkel om die resultate wat in die in vitro studie gekry is, te bevestig. Daar is bewys dat akute DKS-geïnduseerde kardiomiotoksisiteit aanleiding gegee het tot verhoogde apoptose, outofagie inhibisie en verhoogde proteolise via die EPS. Hierdie bevindinge is geassosieer met ‘n verlaging in liggaamsgewig en kardiomiosiet dwarssnit area. Die kardiotoksiese effekte van DKS is insiggewend verminder as autofagiege ïnduseer is met rapamisien. Om saam te vat: Ons data bevestig dat dit moontlik is om die kardiotoksiese effekte van DKS in kanker pasiënte te verminder deur outofagie vlakke te monitor en te kontroleer deur middel van rapamisien behandeling as bykomende terapie.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A utilização de biomarcadores cardioespecíficos vem sendo recomendado como ferramenta útil na monitoração e identificação precoce de lesão cardíaca, em decorrência do potencial de cardiotoxicidade da terapêutica oncologica. O objetivo do presente estudo foi avaliar o nível plasmático do peptídeo natriurético do tipo B (BNP) e da expressão gênica do BNP e outros genes relacionados a sua síntese, a interleucina-6 (IL-6), fator β1 de transformação de crescimento (TGF-β1) e procolágeno tipo I, mediante a associação dos agentes antineoplásicos docetaxel e ciclofosfamida (TC) e da radiação ionizante (IR) no coração de ratas Wistar, 2 meses após o término do tratamento. Para isso, Ratas Wistar (3-4 meses, n=7) foram irradiadas no coração com dose única de 20Gy, em um campo ântero-posterior de 2x2cm2, em acelerador linear com feixe de energia nominal de 6 MeV; outras (n=7) foram tratadas (4 ciclos, com 7 dias de intervalo) com docetaxel (12,5 mg/Kg) e ciclofosfamida (50 mg/Kg) e irradiadas após 7 dias do tratamento quimioterápico. Como controle (n=7), animais não irradiados e não tratados com quimioterápicos. Após 2 meses do fim do tratamento, a eutanásia dos animais foi realizada. Amostras de plasma e tecido cardíaco, ventrículo esquerdo (VE), foram coletadas. Por ensaio ELISA foi quantificada a concentração plasmática de BNP; parte do tecido cardíaco foi fixado, incluído em parafina e cortado em micrótomo, para assinalar a presença de BNP no VE, avaliação qualitativa, pela técnica de imunohistoquímica (IHQ); e a outra parte para a técnica RT-qPCR, onde foram avaliados a expressão relativa de mRNA dos genes do BNP, IL-6, TGF-β1 e procolágeno tipo I. Na IHQ o grupo controle apresentou uma marcação pontual, enquanto que os grupos tratados apresentaram uma marcação mais difusa, sendo que o grupo TC+IR foi o que apresentou maior dispersão na marcação do BNP no tecido cardíaco. Embora não tenha sido observado no ensaio ELISA uma diferença significativa entre as concentrações plasmáticas de BNP dos grupos tratados em relação ao controle, nota-se uma tendência de aumento no grupo TC+IR. Na analise por RT-qPCR, a expressão relativa de BNP foi similar ao apresentado no ELISA. O grupo TC+IR foi o grupo que apresentou maior expressão gênica de BNP, porém a diferença não é significativa em relação ao controle. A única análise em que se obteve diferença na expressão gênica em relação ao controle foi a do gene IL-6 que apresentou expressão reduzida. Todos os demais genes analisados por RT-qPCR apresentaram uma expressão similar ao controle. Assim, os resultados obtidos sugerem que o BNP não se apresentou como um bom biomarcador cardioespecífico para identificação precoce de lesão cardíaca, no período a qual foi avaliado. As ratas Wistar, 2 meses após a submissão do tratamento, não apresentaram um resultado diferenciado em relação ao controle, nos genes TGF-β1 e procolágeno tipo I, sugerindo ausência de um quadro de remodelamento cardíaco. Entretanto, apresentou redução significativa do gene IL-6, no grupo TC+IR, propondo ação anti-inflamatória do BNP, que no mesmo grupo, apresentou uma tendência de aumento em sua expressão gênica. / The use of specific cardiac biomarkers has been recommended as a useful tool in the early identification and monitoring of cardiac injury, due to the potential cardiotoxicity of oncological therapy. The aim of this study is to investigate, analyze and evaluate the reaction of B-type natriuretic peptide (BNP), interleukin-6 (IL-6), transforming growth factor β1 (TGF-β1) and procollagen type I in its expression and release by the association of antineoplastic agents Docetaxel and Cyclophosphamide (TC) and ionizing radiation (IR) in the heart of Wistar rats, 2 months after the end of treatment. For this, Wistar rats (3-4 months, n = 7) were irradiated in the heart with a single dose of 20 Gy, in a anteroposterior field of 2x2cm2 in linear accelerator with nominal energy beam of 6 MeV, other (n = 7) were treated (four cycles with a 7-day interval) with docetaxel (12.5 mg / kg) and Cyclophosphamide (50 mg / kg) and irradiated after 7 days of chemotherapy. As a control (n = 7), non-irradiated animals not treated with chemotherapy. 2 months after the end of treatment, euthanasia was performed. Samples of plasma and heart tissue, left ventricular (LV) were collected. The plasma concentration of BNP was quantified by ELISA, part of the heart tissue was fixed, embedded in paraffin and cut in microtome, to signal the presence of BNP in the LV, qualitative evaluation by immunohistochemistry (IHC), and the other party to technique RT-qPCR were evaluated in the relative expression of mRNA of the genes of BNP, ANP, IL-6, TGF-β1 and procollagen type I. In the control group IHC showed a marking point, while the treated groups showed a more diffuse labeling, and the CT + RT group showed the greatest dispersion. Although it was not observed in the ELISA assay a significant difference between plasma concentrations of BNP treated groups compared to control, there is an increasing trend in the CT + RT group. In the analysis by RT-qPCR, the relative expression of BNP was similar to that shown in the ELISA. The CT + RT group was the group that showed higher gene expression of BNP, but the difference is not significant compared to control. The only analysis that which obtained difference in gene expression compared to control was the IL-6 gene that showed low expression. All other genes analyzed by RT-qPCR showed an expression similar to control. Therefore, the outcome is that BNP did not appear as a good specific cardiac biomarker for early identification of cardiac disease, within 2 months after treatment in Wistar rats, by effect of the action of cardiotoxic selected treatment protocols, since none of their quantitative assessments showed a differentiated result compared to control, however, there was no evidence that, in the meantime, there would be a scenario in development, or advanced of cardiac remodeling.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A utilização de biomarcadores cardioespecíficos vem sendo recomendado como ferramenta útil na monitoração e identificação precoce de lesão cardíaca, em decorrência do potencial de cardiotoxicidade da terapêutica oncologica. O objetivo do presente estudo foi avaliar o nível plasmático do peptídeo natriurético do tipo B (BNP) e da expressão gênica do BNP e outros genes relacionados a sua síntese, a interleucina-6 (IL-6), fator β1 de transformação de crescimento (TGF-β1) e procolágeno tipo I, mediante a associação dos agentes antineoplásicos docetaxel e ciclofosfamida (TC) e da radiação ionizante (IR) no coração de ratas Wistar, 2 meses após o término do tratamento. Para isso, Ratas Wistar (3-4 meses, n=7) foram irradiadas no coração com dose única de 20Gy, em um campo ântero-posterior de 2x2cm2, em acelerador linear com feixe de energia nominal de 6 MeV; outras (n=7) foram tratadas (4 ciclos, com 7 dias de intervalo) com docetaxel (12,5 mg/Kg) e ciclofosfamida (50 mg/Kg) e irradiadas após 7 dias do tratamento quimioterápico. Como controle (n=7), animais não irradiados e não tratados com quimioterápicos. Após 2 meses do fim do tratamento, a eutanásia dos animais foi realizada. Amostras de plasma e tecido cardíaco, ventrículo esquerdo (VE), foram coletadas. Por ensaio ELISA foi quantificada a concentração plasmática de BNP; parte do tecido cardíaco foi fixado, incluído em parafina e cortado em micrótomo, para assinalar a presença de BNP no VE, avaliação qualitativa, pela técnica de imunohistoquímica (IHQ); e a outra parte para a técnica RT-qPCR, onde foram avaliados a expressão relativa de mRNA dos genes do BNP, IL-6, TGF-β1 e procolágeno tipo I. Na IHQ o grupo controle apresentou uma marcação pontual, enquanto que os grupos tratados apresentaram uma marcação mais difusa, sendo que o grupo TC+IR foi o que apresentou maior dispersão na marcação do BNP no tecido cardíaco. Embora não tenha sido observado no ensaio ELISA uma diferença significativa entre as concentrações plasmáticas de BNP dos grupos tratados em relação ao controle, nota-se uma tendência de aumento no grupo TC+IR. Na analise por RT-qPCR, a expressão relativa de BNP foi similar ao apresentado no ELISA. O grupo TC+IR foi o grupo que apresentou maior expressão gênica de BNP, porém a diferença não é significativa em relação ao controle. A única análise em que se obteve diferença na expressão gênica em relação ao controle foi a do gene IL-6 que apresentou expressão reduzida. Todos os demais genes analisados por RT-qPCR apresentaram uma expressão similar ao controle. Assim, os resultados obtidos sugerem que o BNP não se apresentou como um bom biomarcador cardioespecífico para identificação precoce de lesão cardíaca, no período a qual foi avaliado. As ratas Wistar, 2 meses após a submissão do tratamento, não apresentaram um resultado diferenciado em relação ao controle, nos genes TGF-β1 e procolágeno tipo I, sugerindo ausência de um quadro de remodelamento cardíaco. Entretanto, apresentou redução significativa do gene IL-6, no grupo TC+IR, propondo ação anti-inflamatória do BNP, que no mesmo grupo, apresentou uma tendência de aumento em sua expressão gênica. / The use of specific cardiac biomarkers has been recommended as a useful tool in the early identification and monitoring of cardiac injury, due to the potential cardiotoxicity of oncological therapy. The aim of this study is to investigate, analyze and evaluate the reaction of B-type natriuretic peptide (BNP), interleukin-6 (IL-6), transforming growth factor β1 (TGF-β1) and procollagen type I in its expression and release by the association of antineoplastic agents Docetaxel and Cyclophosphamide (TC) and ionizing radiation (IR) in the heart of Wistar rats, 2 months after the end of treatment. For this, Wistar rats (3-4 months, n = 7) were irradiated in the heart with a single dose of 20 Gy, in a anteroposterior field of 2x2cm2 in linear accelerator with nominal energy beam of 6 MeV, other (n = 7) were treated (four cycles with a 7-day interval) with docetaxel (12.5 mg / kg) and Cyclophosphamide (50 mg / kg) and irradiated after 7 days of chemotherapy. As a control (n = 7), non-irradiated animals not treated with chemotherapy. 2 months after the end of treatment, euthanasia was performed. Samples of plasma and heart tissue, left ventricular (LV) were collected. The plasma concentration of BNP was quantified by ELISA, part of the heart tissue was fixed, embedded in paraffin and cut in microtome, to signal the presence of BNP in the LV, qualitative evaluation by immunohistochemistry (IHC), and the other party to technique RT-qPCR were evaluated in the relative expression of mRNA of the genes of BNP, ANP, IL-6, TGF-β1 and procollagen type I. In the control group IHC showed a marking point, while the treated groups showed a more diffuse labeling, and the CT + RT group showed the greatest dispersion. Although it was not observed in the ELISA assay a significant difference between plasma concentrations of BNP treated groups compared to control, there is an increasing trend in the CT + RT group. In the analysis by RT-qPCR, the relative expression of BNP was similar to that shown in the ELISA. The CT + RT group was the group that showed higher gene expression of BNP, but the difference is not significant compared to control. The only analysis that which obtained difference in gene expression compared to control was the IL-6 gene that showed low expression. All other genes analyzed by RT-qPCR showed an expression similar to control. Therefore, the outcome is that BNP did not appear as a good specific cardiac biomarker for early identification of cardiac disease, within 2 months after treatment in Wistar rats, by effect of the action of cardiotoxic selected treatment protocols, since none of their quantitative assessments showed a differentiated result compared to control, however, there was no evidence that, in the meantime, there would be a scenario in development, or advanced of cardiac remodeling.

Made available in DSpace on 2016-08-19T17:37:06Z (GMT). No. of bitstreams: 1 DISSERTACAO_ALCYONE DE OLIVEIRA PAREDES.pdf: 1331038 bytes, checksum: fc7e0955b5d3a1b0cf506e234613229e (MD5) Previous issue date: 2014-11-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The increased incidence of breast cancer in recent years in developed and developing countries has been the main cause of mortality among women aged 40-59 years old. Advances in cancer treatment have increased the quality and survival of patients, however, the adverse effects arising from the therapeutic regimen are extensive and complex, occurring for cardiovascular complications. The Heart Rate Variability (HRV) is a non-invasive parameter, which can be used to identify phenomena related to nerve modulation of the heart, identifying possible changes in this organ. The present study aimed to assess heart rate variability in women with breast carcinoma and treated with doxorrubicin. It was an analytical, cross-sectional study in Maranhense Institute of Oncology, Hospital Aldenora Bello (IMOAB) in the period between March and August 2014 The sample was non-probabilistic type, with 24 female participants, ranging age between 35 to 59 years, divided into two groups, the first experimental group (EG), composed of women with breast carcinoma treatment and control group (CG), composed of women without breast cancer. For data collection, a 12-lead electrocardiogram for the evaluation of heart rate variability, moment to moment where the RR intervals were recorded during the period was used. The participants remained lying at rest in the supine position, and the electrocardiogram was monitored for 15 minutes. RR, and RMSS SDRR indices were used in the time domain (TD) and the LF, HF and LF/HF components in the frequency domain (FD). The data were compiled by BioEstat 5.0 program. No significant differences in variables between the groups were found in demographic and clinical characteristics. The 12 participants in the experimental group were aged between 35 and 59 years, and of these, 66.7% were between 50-59 years old and 33.3% were 40 years or less. There was a reduction of HRV in the DT and DF in the experimental group. Based on the calculation of the individual dose of doxorubicin based on body surface, were found cumulative doses of doxorubicin, above 400 mg/m2 in all experimental group. Early detection of cardiotoxic effects at the beginning or during treatment may help in choosing strategies that enable the reduction of cardiovascular risk. When used in women with breast carcinoma who used doxorubicin, HRV to detect potential heart problems, direct further research and treatment of these changes, thus contributing to improve the prognosis. / O aumento da incidência de câncer de mama nos últimos anos em países desenvolvidos e em desenvolvimento vem sendo responsável pela principal causa de mortalidade entre as mulheres na faixa etária de 40 a 59 anos de idade. Os avanços no tratamento oncológico aumentaram a qualidade e a sobrevida das pacientes, entretanto, os efeitos adversos oriundo do esquema terapêutico são extensos e complexos, com ocorrência para as complicações cardiovasculares. A Variabilidade da Frequência Cardíaca (VFC) é um parâmetro não invasivo, que pode ser utilizada para identificar fenômenos relacionados à modulação nervosa do coração, identificando possíveis alterações nesse órgão. O presente estudo teve como objetivo avaliar a variabilidade da frequência cardíaca em portadoras de carcinoma mamário submetidas a quimioterapia com doxorrubicina. Tratou-se de um estudo analítico e transversal, realizado no Instituto Maranhense de Oncologia, Hospital Aldenora Bello (IMOAB), no período de março a agosto de 2014. A amostra foi por conveniência, com 24 participantes do sexo feminino, na faixa etária entre 35 a 59 anos, divididas em dois grupos, sendo o primeiro grupo experimental (GE), composto por mulheres portadoras de carcinoma mamário em tratamento e o grupo controle (GC), composto por mulheres sem carcinoma mamário. Para a coleta de dados foi utilizado um eletrocardiograma de 12 derivações para a avaliação da variabilidade da frequência cardíaca, onde foram registrados momento a momento os intervalos R-R durante o período de repouso. As participantes permaneceram deitadas em repouso na posição supina, e o eletrocardiograma foi monitorado por 15 minutos. Foram utilizados os índices RR, SDRR e RMSS no domínio do tempo (DT) e os componentes LF, HF e LF/HF no domínio da frequência (DF). Os dados obtidos foram compilados pelo programa BioEstat 5.0. Não foram encontradas diferenças significativas das variáveis analisadas entre os grupos em relação às características clínicodemográficas. As 12 participantes do grupo experimental estavam na faixa etária entre 35 a 59 anos, sendo que destas, 66,7% tinham entre 50 a 59 anos de idade e 33,3% tinham 40 anos ou menos. Houve redução da VFC no DT e DF no grupo experimental. Baseando-se no cálculo da dose individual de doxorrubicina com base na superfície corporal, foram encontradas doses cumulativas de doxorrubicina, acima de 400mg/m2 em todas as participantes do grupo experimental. A detecção precoce dos efeitos cardiotóxicos no início ou no decorrer do tratamento, pode auxiliar na escolha de estratégias que possibilitem a redução do risco cardiovascular. Quando utilizada em portadoras de carcinoma mamário que fizeram uso de doxorrubicina, a VFC permitirá detectar possíveis alterações cardíacas, direcionar o aprofundamento da investigação e tratamento dessas alterações, contribuindo assim na melhora do prognóstico.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Pavla Martinková Supervisor: PharmDr. Anna Jirkovská, PhD. Title of diploma thesis: Antiproliferative activity of novel dexrazoxane analogues and their effect on antitumor effectiveness of anthracyclines Athracycline antibiotics (such as daunorubicin, doxorubicin or epirubicin) belong to the most common terapeutics of both solid tumors and hematological malignities. Unfortunately the serious and life-threatening adverse effect cardiotoxicity compromises their clinical usefulness. The only approved protection against anthracycline cardiotoxicity so far is dexrazoxane. Despite the outstanding cardioprotective ability, dexrazoxane use is very limited mainly due to its possible side effects. So we were directed towards synthesis of dexrazoxane analogues with better pharmacological properties. The aim of this diploma thesis was to assess the antiproliferative activity of novel analogues of both dexrazoxane (MK-15 and ES-5) and ADR-925 (JR-159 and KH- TA4) and their influence on the antiproliferative effectiveness of anthracyclines. Moreover, we aimed to study their chelating properties and their inhibition of the topoisomerase II in solution. We tested the antiproliferative activity of...

The aim of this study was to investigate the mode of action of pavetamine on rat cardiomyocytes. Pavetamine is the causative agent of gousiekte (“quick-disease”), a disease of ruminants characterized by acute heart failure following ingestion of certain rubiaceous plants. Two in vitro rat cardiomyocyte models were utilized in this study, namely the rat embryonic cardiac cell line, H9c2, and primary neonatal rat cardiomyocytes. Cytotoxicity of pavetamine was evaluated in H9c2 cells using the MTT and LDH release assays. The eventual cell death of H9c2 cells was due to necrosis, with LDH release into the culture medium after exposure to pavetamine for 72 h. Pavetamine did not induce apoptosis, as the typical features of apoptosis were not observed. Electron microscopy was employed to study ultrastructural alterations caused by pavetamine in H9c2 cells. The mitochondria and sarcoplasmic reticula showed abnormalities after 48 h exposure of the cells to pavetamine. Abundant secondary lysosomes with electron dense material were present in treated cells. Numerous vacuoles were also present in treated cells, indicative of autophagy. During this exposure time, the nuclei appeared normal, with no chromatin condensation as would be expected for apoptosis. Abnormalities in the morphology of the nuclei were only evident after 72 h exposure. The nuclei became fragmented and plasma membrane blebbing occurred. The mitochondrial membrane potential was investigated with a fluorescent probe, which demonstrated that pavetamine caused significant hyperpolarization of the mitochondrial membrane, in contrast to the depolarization caused by apoptotic inducers. Pavetamine did not cause opening of the mitochondrial permeability transition pore, because cyclosporine A, which is an inhibitor of the mitochondrial permeability transition pore, did not reduce the cytotoxicity of pavetamine significantly. Fluorescent probes were used to investigate subcellular changes induced by pavetamine in H9c2 cells. The mitochondria and sarcoplasmic reticula showed abnormal features compared to the control cells, which is consistent with the electron microscopy studies. The lysosomes of treated cells were more abundant and enlarged. The activity of cytosolic hexosaminidase was nearly three times higher in the treated cells than in the control cells, which suggested increased lysosomal membrane permeability. The activity of acid phosphatase was also increased in comparison to the control cells. In addition, the organization of the cytoskeletal F-actin of treated cells was severely affected by pavetamine. Rat neonatal cardiomyocytes were labelled with antibodies to detect the three major contractile proteins (titin, actin and myosin) and cytoskeletal proteins (F-actin, desmin and β-tubulin). Cells treated with pavetamine had degraded myosin and titin, with altered morphology of sarcomeric actin. Vacuoles appeared in the β-tubulin network, but the appearance of desmin was normal. F-actin was severely disrupted in cardiomyocytes treated with pavetamine and was degraded or even absent in treated cells. Ultrastructurally, the sarcomeres of rat neonatal cardiomyocytes exposed to pavetamine were disorganized and disengaged from the Z-lines, which can also be observed in the hearts of ruminants that have died of gousiekte. It is concluded that the pathological alteration to the major contractile and cytoskeleton proteins caused by pavetamine could explain the cardiac dysfunction that characterizes gousiekte. F-actin is involved in protein synthesis and therefore can play a role in the inhibition of protein synthesis in the myocardium of ruminants suffering from gousiekte. Apart from inhibition of protein synthesis in the heart, there is also increased degradation of cardiac proteins in an animal with gousiekte. The mitochondrial damage will lead to an energy deficiency and possibly to generation of reactive oxygen species. The sarcoplasmic reticula are involved in protein synthesis and any damage to them will affect protein synthesis, folding and post-translational modifications. This will activate the unfolded protein response (UPR) and sarcoplasmic reticula-associated protein degradation (ERAD). If the oxidizing environment of the sarcoplasmic reticula is disturbed, it will activate the ubiquitin-proteasome pathway (UPP) to clear aggregated and misfolded proteins. Lastly, the mitochondria, sarcoplasmic reticula and F-actin are involved in calcium homeostasis. Any damage to these organelles will have a profound influence on calcium flux in the heart and will further contribute to the contractile dysfunction that characterizes gousiekte. / Thesis (PhD)--University of Pretoria, 2010. / Paraclinical Sciences / unrestricted

Introducción: Los índices linfocitarios, neutrófilo-linfocito (INL), monocito- linfocito (IML) y plaqueta-linfocito (IPL) son marcadores inflamatorios de valor pronóstico en enfermedades cardiovasculares. Nuestro objetivo es evaluar la asociación entre dichos índices y el desarrollo de cardiotoxicidad en pacientes con cáncer de mama que recibieron quimioterapia entre los años 2016-2019. Métodos: Cohorte retrospectiva, basada en la revisión de resultados ecocardiográficos y de laboratorio en mujeres ≥18 años con diagnóstico confirmado de cáncer de mama que recibieron quimioterapia en una clínica peruana entre 2016-2019. Cardiotoxicidad durante la quimioterapia se definió como reducción de la fracción de eyección del ventrículo izquierdo ≥10%, reducción del 20% del strain miocárdico, o aparición de una nueva disfunción ventricular izquierda. El INL se calculó como el cociente entre el número absoluto de neutrófilos y linfocitos obtenidas en el hemograma más cercano antes de iniciar quimioterapia. De forma similar, se calcularon el IML e IPL. La magnitud de asociación se evaluó usando Riesgos Relativos (RR) crudos y ajustados con intervalos de confianza al 95% (IC95%), obtenidos mediante regresión de Poisson con varianza robusta. Resultados: 418 mujeres con cáncer de mama recibieron quimioterapia en la clínica participante y tuvieron ecocardiografías. De ellas, se excluyeron 176 por no tener ecocardiografías control. En los 242 participantes, la media de edad fue 56.5 (DE 12.3). La incidencia acumulada de cardiotoxicidad fue 12.4% durante la quimioterapia. En el análisis bivariado se encontró asociación con Hipertensión y Diabetes. No se encontró asociación con ninguno de los índices linfocitarios. En el análisis ajustado por edad y presencia de comorbilidades, hubo un RR de 1.06 (IC95% 0.95-1.19) por cada punto adicional del INL. Conclusiones: No se encontró asociación entre desarrollo de cardiotoxicidad con los índices linfocitarios. Se requieren más estudios con mayor número de participantes para determinar si efectivamente no existe asociación con los índices estudiados. / Background: Lymphocyte indices, neutrophil-lymphocyte (NLI), monocyte- lymphocyte (MLI) and platelet-lymphocyte (PLI) are inflammatory markers of prognostic value on cardiovascular diseases. Our goal is to evaluate the relation between the lymphocyte indices and the development of cardiotoxicity on patients with breast cancer who received chemotherapy between 2016-2019. Methods: Retrospective cohort study based on the revision of echocardiogram and labs results among women ≥18 years old with confirmed breast cancer diagnosis who received chemotherapy in a Peruvian clinic between 2016-2019. Cardiotoxicity during chemotherapy was defined as ≥10% reduction in the left ventricle ejection fraction, ≥20% reduction of myocardial strain, or new left ventricle dysfunction. NLI was calculated as the quotient between the absolute neutrophil and lymphocyte counts in the most recent complete blood count before chemotherapy. MLI and PLI were calculated similarly. Association was evaluated by means of crude and adjusted Relative Risks (RR) with their 95% confidence intervals (95%CI), using Poisson regression with robust variance. Results: 418 women with breast cancer received chemotherapy and had echocardiographic evaluations. 176 were excluded because they lacked follow- up echocardiograms. From the 242 participants, the mean age was 56.5 (SD 12.3). Cumulative cardiotoxicity incidence was 12.4% during chemotherapy. There was association with hypertension and diabetes in the bivariate analysis. No association with any of the Lymphocyte indices was found in the adjusted models. The RR for each additional NLI point was 1.06 (95%CI 0.95-1.19). Conclusions: There was no association between cardiotoxicity during the chemotherapy with the Lymphocyte indices. More studies with more participants are needed in order to really determine if there is no association with the studied indices. / Tesis

The current studies were designed to explore the protective effects of pifithrin-α and melatonin against doxorubicin-induced cardiotoxicity. Doxorubicin was injected at a dose of 22.5 mg/kg (i.p.) in mice to induce cardiotoxic effects. Meanwhile, doxorubicin caused a significant increase of cardiac cell apoptosis following injection (14.2 ± 1.1% for doxorubicin-5 d vs. 1.8 ± 0.12% for control, P < 0.01). Ribonuclease protection assays and Western blot analyses revealed that doxorubicin upregulated the p53-dependent genes Bax, BclxL, and MDM2 at least 2-fold. p53 was phosphorylated at Ser 15 in mouse hearts 1 h following doxorubicin injection, and p38 and ERK1/2 MAPKs mediated the phosphorylation of p53. In addition, caspases-3 and -9 were activated 24 h after doxorubicin injection. A p53 inhibitor, pifithrin-α, inhibited doxorubicin-induced apoptosis when administered at a dose of 2.2 mg/kg. Pifithrin-α abolished p53 transactivation activity, but did not influence doxorubicin-induced phosphorylation at Ser 15. By effectively inhibiting the expression of p53-dependent genes, pifithrin-α blocked doxorubicin-induced activation of caspases-3 and -9, thereby preventing cardiac apoptosis. In addition, pifithrin-α attenuated doxorubicin-induced structural and functional damages, without diminishing its anti-tumor efficacy on p53-null PC-3 cancer cells. The protective effects of melatonin and its metabolite 6-hydroxymelatonin on doxorubicin-induced cardiac dysfunction were evaluated in an isolated perfused mouse hearts and in vivo doxorubicin-treated mice. While perfusion of mouse hearts with 5 μM doxorubicin for 60 min resulted in a 50% suppression of HRxLVDP and a 50% reduction of coronary flow, pre-exposure of hearts to 1 μM melatonin or 6-hydroxymelatonin eased the cardiac dysfunction. In addition, administration of melatonin or 6-hydroxymelatonin (2 mg/kg/d) significantly attenuated doxorubicin-induced cardiac dysfunction, myocardial lesions, and cardiac cell apoptosis. Melatonin and 6-hydroxymelatonin significantly improved the survival rate of doxorubicin-treated mice. Another melatonin analog, 8-methoxy-2-propionamidotetralin, did not show any convincing protection on either animal survival or on in vitro cardiac function, presumably due to its lack of free radical-scavenging activity. Finally, neither melatonin nor 6-hydroxymelatonin compromised the anti-tumor activity of doxorubicin in cultured PC-3 cells. These studies suggest that pifithrin-α and melatonin have significant therapeutic potential for patients suffering doxorubicin-induced cardiotoxicity.

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