Spermatogenesis is a complex process of male germ cell development from diploid spermatogonia to haploid fertile spermatozoa. Apoptosis plays a vital role in limiting cell numbers and eliminating defective germ cells. This requires novel gene products, and precise and well-coordinated programmes of gene expression. It is therefore possible that a disruption of transcription factor function would significantly impact germ cell development. The present work was undertaken to use Staput separation followed by culture of purified germ cells of rodent testis since mammalian spermatogenesis cannot yet be recreated in vitro. Specificity of separation was assessed using immunocytochemistry to identify spermatogonia, spermatocytes and spermatids. The genotoxins H2O2, doxorubicin, N-ethyl-N-nitrosourea, N-methyl-N-nitrosourea, 6-mercaptopurine, 5-bromodeoxyuridine, methyl methanesulphonate and ethyl methanesulphonate were investigated. Cells were cultured and treated with different concentrations for each agent. DNA damage and apoptosis were measured by Comet and TUNEL assay respectively. Up-regulation of expression of the transcription factors Tbpl1, FHL5 and Gtf2a1l that are important post-meiotically, were examined using RT- PCR and qPCR. Protein production was evaluated using Western blotting. Tbpl1, FHL5 and Gtf2a1l were cloned in-frame into the inducible expression vector pET/100-TOPO. The recombinant clones were induced and successful expression of the proteins in E. coli was confirmed by SDS-PAGE and Western blotting. The recombinant clones obtained were used to demonstrate genotoxin induced impairment of gene expression. Thus, Staput-isolated rodent testicular germ cells seem to be a suitable model to study genotoxicity in vitro yielding result comparable to those reported in vivo. Furthermore, the work shows that genotoxins can impair gene expression.

Hu Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 95-122). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract (in English) --- p.ii / Abstract (in Chinese) --- p.iv / Table of contents --- p.vi / List of figures --- p.x / List of tables --- p.xi / Abbreviations --- p.vii / Chapter CHAPTER ONE: --- INTRODUCATION --- p.1 / Chapter 1.1 --- Hepatocellular Carcinoma --- p.2 / Chapter 1.1.1. --- Epidemiology of HCC --- p.2 / Chapter 1.1.2. --- The major risk factors --- p.2 / Chapter 1.1.3. --- Management of HCC --- p.3 / Chapter 1.2 --- Mechanisms of multidrug resistance (MDR) in cancer cells --- p.4 / Chapter 1.2.1. --- Major mechanisms in reduced drug accumulation --- p.5 / Chapter 1.2.1.1. --- P-glycoprotein (P-gp) --- p.6 / Chapter 1.2.1.2. --- Multidrug Resistance-associated Protein (MRP) --- p.7 / Chapter 1.2.1.3. --- Other effluxes --- p.8 / Chapter 1.2.2. --- Inhibition of apoptotic signaling pathways --- p.11 / Chapter 1.2.2.1. --- TP53 and multidrug resistance --- p.11 / Chapter 1.2.2.2. --- Anti-oncogene PTEN and drug resistance --- p.13 / Chapter 1.2.2.3. --- Influence of BCL2 family on drug resistance --- p.14 / Chapter 1.3 --- The chemotherapeutic agent of doxorubicin --- p.15 / Chapter 1.4 --- Aims of study --- p.18 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.20 / Chapter 2.1 --- Cell culture --- p.21 / Chapter 2.1.1 --- Cell lines and cell culture --- p.21 / Chapter 2.1.2 --- Subculture --- p.23 / Chapter 2.1.3 --- Cryopreservation --- p.23 / Chapter 2.1.4 --- Recovery of cryopreserved culture --- p.24 / Chapter 2.1.5 --- Cell number counting --- p.24 / Chapter 2.2 --- MTT experiments --- p.26 / Chapter 2.2.1 --- Determination of cell seeding density --- p.26 / Chapter 2.2.2 --- Cytotoxic assay --- p.27 / Chapter 2.3 --- Spectral Karytyping (SKY) --- p.27 / Chapter 2.3.1 --- Pretreatment of chromosome slides for SKY --- p.28 / Chapter 2.3.2 --- Hybridization --- p.28 / Chapter 2.3.3 --- Detection --- p.29 / Chapter 2.4 --- Positional expression profiling --- p.30 / Chapter 2.4.1 --- RNA extraction --- p.32 / Chapter 2.4.2 --- Reverse transcription and cDNA labling --- p.34 / Chapter 2.4.3 --- Probe purification and hybridization --- p.34 / Chapter 2.4.4 --- Image acquisition and data analysis --- p.35 / Chapter 2. 5 --- Quantitative RT-PCR --- p.37 / Chapter 2.5.1 --- RNA extraction --- p.37 / Chapter 2.5.2 --- Primer design --- p.37 / Chapter 2.5.3 --- Reverse transcription --- p.37 / Chapter 2.5.4 --- Quantitative PCR --- p.39 / Chapter 2.6. --- Statistical analysis --- p.40 / Chapter CHAPTER 3 --- RESULTS --- p.43 / Introduction --- p.44 / Chapter 3.1 --- Doxorubicin resistance in HCC cell lines --- p.44 / Chapter 3.2 --- Candidate drug resistance genes --- p.56 / Chapter 3.3 --- The roles of chromosomal instability --- p.58 / Chapter 3.4 --- Candidate resistance genes identified in chromosome 10 --- p.69 / Chapter CHAPTER 4 --- DISCUSSION --- p.75 / Introduction --- p.76 / Chapter 4.1 --- In vitro cell models facilitate drug resistance investigations --- p.11 / Chapter 4.2 --- Aneuploidy and DX resistance --- p.78 / Chapter 4.3 --- The role of known resistance genes on chromosome 10 --- p.79 / Chapter 4.4 --- Identification of novel DX resistance genes on chromosome 10 --- p.80 / Chapter 4.5 --- Common drug resistance genes --- p.83 / Chapter 4.5.1. --- The roles of classical drug resistance --- p.85 / Chapter 4.5.2. --- Inhibition of apoptosis and deregulation of cell cycle --- p.86 / Chapter CHAPTER 5 --- PROPOSED FUTURE STUDIES --- p.90 / Chapter 5.1. --- Validate significant in vitro findings by clinical trials --- p.91 / Chapter 5.2. --- Molecular mechanisms in inactivation of ECHS1 in resistant cells --- p.92 / Chapter 5.3. --- Future utilization of cDNA microarray data --- p.93 / REFERENCES --- p.95 / PUBLICATION --- p.122

Orientador: Daisy Maria Fávero Salvadori / Resumo: A cardiotoxicidade induzida pela doxorrubicina (DOX), antraciclina isolada da actinobacteria Streptomyces peucetius e amplamente utilizada na terapia antineoplásica, corresponde a um dos mais importantes eventos patofisiológicos que limitam sua aplicação clínica. No entanto, não são completamente conhecidos todos os mecanismos envolvidos nessa toxicidade, o que diminui as possiblidades de intervenção e a redução dos efeitos colaterais para os pacientes sob tratamento. Uma das hipósteses é que os aldeídos gerados pela ação da DOX atuam sobre membranas mitocondriais, alterando o estado redox e formando adutos com proteínas, os quais prejudicam o correto funcionamento da organela. Atividades deletérias da DOX sobre outros componentes celulares, como, por exemplo, os ácidos ribonucleicos, são, também, possíveis mecanismos de toxicidade do antineoplásico. Várias estratégias têm sido utilizadas para minimizar os efeitos adversos da DOX. Uma delas, é a busca por compostos que possam proteger as células da ação citotóxica. Nesse sentido, a Alda-1, pertencente ao grupo das chaperonas e agonista da enzima aldeído desidrogenase mitocondrial (ALDH2), vem sendo testada com o objetivo de reduzir os efeitos adversos dos metabólitos e radicais gerados pelo antineoplásico. Para investigar outros possíveis mecanismos de ação da DOX e o efeito cardiprotetor da Alda-1, este estudo foi delineado utilizando duas abordagens distintas: experimentos in vivo, com ratos Wistar machos submetidos a trata... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The cardiotoxicity induced by doxorubicin (DOX), anthracycline isolated from the actinobacteria Streptomyces peucetius, and widely used as an antineoplastic drug, is one of the most important pathophysiological events that limit its clinical application. However, all the mechanisms involved in this toxicity are not fully understood. One hypothesis is that the aldehydes generated by DOX act on mitochondrial membranes, modifying the redox state and proting adducts with proteins. DOX activities on other cellular components, such as ribonucleic acids, are also possible mechanisms of toxicity. Several strategies have been used to reduce the DOX adverse effects. One of them is the identification of compounds that can protect cells against cytotoxic. Alda-1, which belongs to a group of chaperones and is an agonist of the mitochondrial aldehyde dehydrogenase (ALDH2), has been tested to reduce the adverse effects of metabolites and radicals generated by DOX. To investigate other possible DOX mechanisms of action, and the cardioprotective activity of Alda-1, this study was designed using two different approaches: in vivo, with male Wistar rats submitted to acute and chronic treatments; and, in vitro, in mice fibroblasts and cybrids with ND5 (gene that encodes the mitochondrial Complex I subunit) mitochondrial gene heteroplasmy. The expression profiling of genes related to beta oxidation pathways, Bax, Bcl-2, C1QBP, ALDH2 and miR34a microRNA (ALDH2 expression regulator), and the lipoper... (Complete abstract click electronic access below) / Doutor

nÃo hà / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / IntroduÃÃo: A Doxorubicina (DOX), antineoplÃsico antracÃclico, à largamente utilizada no tratamento dos mais diversos tipos de tumores sÃlidos e neoplasias hematolÃgicas. A Cardiotoxicidade provocada pelo uso de antibiÃticos antracÃclicos tem sido observada hà algumas dÃcadas como fator agravante e limitante do uso terapÃutico da DOX. Apesar do conhecimento de inÃmeros fatores que podem mediar a induÃÃo da cardiotoxicidade pela DOX, os mecanismos fisiopatolÃgicos continuam nÃo esclarecidos. Objetivo: Avaliar o efeito da amifostina e glutationa na cardiotoxicidade aguda induzida por doxorubicina e estudar a morfologia do tecido cardÃaco de camundongos tratados com doxorubicina por microscopia de forÃa atÃmica. Material e MÃtodos: Camundongos C57BL/6 fÃmeas (n=8) foram tratados com doxorubicina (25mg/Kg i.p.) ou salina (0,2mL i.p.) e sacrificados 96 horas apÃs tratamento. Outro grupo experimental foi tratado com amifostina (AMF 25, 50 e 100 mg/Kg s.c.), glutationa (GLT 125, 250 e 500mg/Kg s.c.) ou salina 30 mim antes da injeÃÃo de doxorubicina, no caso da glutationa a administraÃÃo foi diÃria atà o dia do sacrifÃcio. Os parÃmetros analisados foram: eletrocardiograma, Ãndices cardÃaco e esplÃnico, dosagem de grupos sulfidrilas nÃo protÃicos, dosagem de CK e CK-MB, parÃmetros histolÃgicos, dosagem por ELISA de TNF-&#61537;, IL-1&#61538;&#61484; expressÃo por imunohistoquÃmica de TNF-&#61537;, IL-1&#61538;, iNOS, apoptose e anÃlise por microscopia de forÃa atÃmica. Resultados: O tratamento com AMF nas doses de 50 e 100mg/Kg e GLT 250 e 500 mg/Kg foi capaz de aumentar a percentagem de sobrevivÃncia dos animais que foram submetidos a cardiotoxicidade aguda induzida por DOX (25 mg/Kg) quando comparados com o grupo injetado somente com DOX. A AMF e GLT tambÃm foram capazes de prevenir, em comparaÃÃo ao grupo DOX (p<0,05), as alteraÃÃes nos valores eletrocardiogrÃficos, (aumento do QRS e QTc e diminuiÃÃo da amplitude de R), as alteraÃÃes nos Ãndices cardÃacos e esplÃnicos, a elevaÃÃo dos nÃveis sÃricos das enzimas CK e CK-MB, a reduÃÃo dos nÃveis de grupos sulfidrilas nÃo protÃicos no tecido cardÃaco e as alteraÃÃes histolÃgicas (degeneraÃÃo hidrÃpica e vacuolizaÃÃo, focos de hialinizaÃÃo de fibras cardÃacas, picnose e necrose) induzidas pela DOX (25mg/Kg). A DOX induziu aumento da marcaÃÃo imunohistoquÃmica para cÃlulas apoptÃticas e expressÃo de iNOS e diminuiu a expressÃo de TNF-&#61537;. A AMF foi capaz de prevenir estas alteraÃÃes, sendo esta prevenÃÃo apenas discreta para a expressÃo de TNF-&#61537;. A microscopia de forÃa atÃmica revelou alteraÃÃes morfolÃgicas nÃo vistas pela microscopia Ãptica e mostrou ser uma ferramenta valiosa na avaliaÃÃo de efeitos de drogas. ConclusÃo: Nossos resultados sugerem o efeito citoprotetor da amifostina pelo aumento da atividade da glutationa peroxidase no tecido cardÃaco e que esta se mostra tÃo eficiente quanto a droga de referencia dexrazoxane. A utilizaÃÃo da microscopia atÃmica introduz uma ferramenta de anÃlise comparativa em escala nanomÃtrica, tornando possÃvel observar a destruiÃÃo membranar cardÃaco condizente com dano oxidativo. / IntroduÃÃo: A Doxorubicina (DOX), antineoplÃsico antracÃclico, à largamente utilizada no tratamento dos mais diversos tipos de tumores sÃlidos e neoplasias hematolÃgicas. A Cardiotoxicidade provocada pelo uso de antibiÃticos antracÃclicos tem sido observada hà algumas dÃcadas como fator agravante e limitante do uso terapÃutico da DOX. Apesar do conhecimento de inÃmeros fatores que podem mediar a induÃÃo da cardiotoxicidade pela DOX, os mecanismos fisiopatolÃgicos continuam nÃo esclarecidos. Objetivo: Avaliar o efeito da amifostina e glutationa na cardiotoxicidade aguda induzida por doxorubicina e estudar a morfologia do tecido cardÃaco de camundongos tratados com doxorubicina por microscopia de forÃa atÃmica. Material e MÃtodos: Camundongos C57BL/6 fÃmeas (n=8) foram tratados com doxorubicina (25mg/Kg i.p.) ou salina (0,2mL i.p.) e sacrificados 96 horas apÃs tratamento. Outro grupo experimental foi tratado com amifostina (AMF 25, 50 e 100 mg/Kg s.c.), glutationa (GLT 125, 250 e 500mg/Kg s.c.) ou salina 30 mim antes da injeÃÃo de doxorubicina, no caso da glutationa a administraÃÃo foi diÃria atà o dia do sacrifÃcio. Os parÃmetros analisados foram: eletrocardiograma, Ãndices cardÃaco e esplÃnico, dosagem de grupos sulfidrilas nÃo protÃicos, dosagem de CK e CK-MB, parÃmetros histolÃgicos, dosagem por ELISA de TNF-&#61537;, IL-1&#61538;&#61484; expressÃo por imunohistoquÃmica de TNF-&#61537;, IL-1&#61538;, iNOS, apoptose e anÃlise por microscopia de forÃa atÃmica. Resultados: O tratamento com AMF nas doses de 50 e 100mg/Kg e GLT 250 e 500 mg/Kg foi capaz de aumentar a percentagem de sobrevivÃncia dos animais que foram submetidos a cardiotoxicidade aguda induzida por DOX (25 mg/Kg) quando comparados com o grupo injetado somente com DOX. A AMF e GLT tambÃm foram capazes de prevenir, em comparaÃÃo ao grupo DOX (p<0,05), as alteraÃÃes nos valores eletrocardiogrÃficos, (aumento do QRS e QTc e diminuiÃÃo da amplitude de R), as alteraÃÃes nos Ãndices cardÃacos e esplÃnicos, a elevaÃÃo dos nÃveis sÃricos das enzimas CK e CK-MB, a reduÃÃo dos nÃveis de grupos sulfidrilas nÃo protÃicos no tecido cardÃaco e as alteraÃÃes histolÃgicas (degeneraÃÃo hidrÃpica e vacuolizaÃÃo, focos de hialinizaÃÃo de fibras cardÃacas, picnose e necrose) induzidas pela DOX (25mg/Kg). A DOX induziu aumento da marcaÃÃo imunohistoquÃmica para cÃlulas apoptÃticas e expressÃo de iNOS e diminuiu a expressÃo de TNF-&#61537;. A AMF foi capaz de prevenir estas alteraÃÃes, sendo esta prevenÃÃo apenas discreta para a expressÃo de TNF-&#61537;. A microscopia de forÃa atÃmica revelou alteraÃÃes morfolÃgicas nÃo vistas pela microscopia Ãptica e mostrou ser uma ferramenta valiosa na avaliaÃÃo de efeitos de drogas. ConclusÃo: Nossos resultados sugerem o efeito citoprotetor da amifostina pelo aumento da atividade da glutationa peroxidase no tecido cardÃaco e que esta se mostra tÃo eficiente quanto a droga de referencia dexrazoxane. A utilizaÃÃo da microscopia atÃmica introduz uma ferramenta de anÃlise comparativa em escala nanomÃtrica, tornando possÃvel observar a destruiÃÃo membranar cardÃaco condizente com dano oxidativo. / Introduction: Doxorubicin (DOX) is an antineoplasic anthracyclic agent used on the treatment of several solid tumors and hematological cancers. DOX-induced cardiotoxicity has been studied for decades as a limiting factor on the anticancer therapy with this drug. Despite the current knowledge concerning the mechanisms of DOX-induced cardotoxicity, its pathophysiology is still not clear. Purpose: To evaluate of the amifostine and glutathione citoprotective effect on the DOX-induced acute cardiotoxicity and to study the morphology of cardiac tissue through the use of atomic force microscopy as a tool. Materials and Methods: C57BL/6 female mice were treated with doxorubicin (25mg/Kg i.p.) or saline (0,2mL i.p.) and sacrificed 96 hours after treatment. In another experimental setting, mice were given amifostine (AMF 25, 50 e 100 mg/Kg s.c.), glutathione (GLT 125, 250 e 500mg/Kg s.c.) or vehicle, 30 mim before the administration of DOX, except glutathione that was injected daily. Analitical parameters included: electrocardiograms, cardiac and spleen indices, non protein suphidrils groups levels, CK and CK-MB cardiac enzymes levels, histological analysis, cardiac levels of (TNF-&#61537;, IL-1&#61538;, iNOS) determined by ELISA, immunohistochemistry for TNF-&#61537;, IL-1&#61538;, iNOS, apoptosis and atomic force microscopy tissue analysis. Results: AMF (100mg/Kg) and GLT (250 e 500 mg/Kg) treatments were able of improve the survival rate of animals in spite of the injection of DOX (25 mg/Kg) in comparison to DOX-treated only group (p<0.05). A AMF e GLT were also able to prevent electrocardiographic changes (rising of QRS e QTc and reduced R amplitude), changes in the cardiac and spleen indices, the augmentation of blood levels of CK e CK-MB, reduction of non proteic suphidrils groups levels, and histological changes induced by DOX (25mg/Kg). DOX induced the augmentation of the immunostaining for apoptotic cells and iNOS what was prevented by the administration of amifostine. The atomic force microscopy reveals morphological changes on the tissue organizational structure which is not possible to be observed through optical microscopy. Conclusion: Our results suggest that the amifostine citoprotective effect on DOX-induced acute cardiotoxicity is due the rising of glutathione peroxidase activity in the cardiac tissue. The citoprotective effect of amifostine is as efficient as the reference drug dexrazoxane. The use of atomic force microscopy as a new pharmacological tool for comparative analysis in nanometric scale allow us to observe DOX-induced membrane destruction what is suggestive of oxidative stress process. / Introduction: Doxorubicin (DOX) is an antineoplasic anthracyclic agent used on the treatment of several solid tumors and hematological cancers. DOX-induced cardiotoxicity has been studied for decades as a limiting factor on the anticancer therapy with this drug. Despite the current knowledge concerning the mechanisms of DOX-induced cardotoxicity, its pathophysiology is still not clear. Purpose: To evaluate of the amifostine and glutathione citoprotective effect on the DOX-induced acute cardiotoxicity and to study the morphology of cardiac tissue through the use of atomic force microscopy as a tool. Materials and Methods: C57BL/6 female mice were treated with doxorubicin (25mg/Kg i.p.) or saline (0,2mL i.p.) and sacrificed 96 hours after treatment. In another experimental setting, mice were given amifostine (AMF 25, 50 e 100 mg/Kg s.c.), glutathione (GLT 125, 250 e 500mg/Kg s.c.) or vehicle, 30 mim before the administration of DOX, except glutathione that was injected daily. Analitical parameters included: electrocardiograms, cardiac and spleen indices, non protein suphidrils groups levels, CK and CK-MB cardiac enzymes levels, histological analysis, cardiac levels of (TNF-&#61537;, IL-1&#61538;, iNOS) determined by ELISA, immunohistochemistry for TNF-&#61537;, IL-1&#61538;, iNOS, apoptosis and atomic force microscopy tissue analysis. Results: AMF (100mg/Kg) and GLT (250 e 500 mg/Kg) treatments were able of improve the survival rate of animals in spite of the injection of DOX (25 mg/Kg) in comparison to DOX-treated only group (p<0.05). A AMF e GLT were also able to prevent electrocardiographic changes (rising of QRS e QTc and reduced R amplitude), changes in the cardiac and spleen indices, the augmentation of blood levels of CK e CK-MB, reduction of non proteic suphidrils groups levels, and histological changes induced by DOX (25mg/Kg). DOX induced the augmentation of the immunostaining for apoptotic cells and iNOS what was prevented by the administration of amifostine. The atomic force microscopy reveals morphological changes on the tissue organizational structure which is not possible to be observed through optical microscopy. Conclusion: Our results suggest that the amifostine citoprotective effect on DOX-induced acute cardiotoxicity is due the rising of glutathione peroxidase activity in the cardiac tissue. The citoprotective effect of amifostine is as efficient as the reference drug dexrazoxane. The use of atomic force microscopy as a new pharmacological tool for comparative analysis in nanometric scale allow us to observe DOX-induced membrane destruction what is suggestive of oxidative stress process.

Les prokinéticines sont des hormones angiogéniques qui exercent leurs fonctions biologiques par l’intermédiaire de deux récepteurs couplés aux protéines G : PKR1 et PKR2. PKR1 a été révélé comme crucial dans l’homéostasie cardiovasculaire. L’objectif de ce projet de thèse était de développer un nouvel agoniste non–peptidique à PKR1 pour la cardioprotection et la régénération cardiaque. Les premiers résultats ont permis de caractériser le premier ligand spécifique à PKR1 : IS20. L’étude in vivo a démontré qu’IS20 est capable de prévenir les lésions après induction d’un infarctus du myocarde chez la souris. Ce composé améliore les fonctions cardiaques en activant la prolifération de cellules progénitrices cardiaques et la néovascularisation (Gasser et al, PlosOne, 2015). Dans une deuxième étude, nous avons évalué le potentiel cardioprotecteur d’IS20 face à la toxicité induite par la doxorubicine (DOX), un anticancéreux de la famille des anthracyclines très efficace mais cardiotoxique. Les résultats montrent qu’IS20 atténue l’apoptose des cardiomyocytes H9c2 et des cellules progénitrices humaines de types EPDC, induite par la doxorubicine, sans affecter la cytotoxicité de la doxorubicine sur les cellules cancéreuses. In vivo, le traitement par IS20 atténue la diminution de la prolifération provoquée par la doxorubicine dans un modèle de cardiotoxicité juvénile. Dans un modèle de cardiotoxicité chronique, IS20 maintient l’intégrité cellulaire et tissulaire des vaisseaux et protège des défaillances produites par DOX. Par ses effets cytoprotecteurs des cardiomyocytes et des cellules progénitrices cardiaques, l’IS20 présente un potentiel thérapeutique prometteur pour protéger les patients cancéreux des effets cardiotoxiques des anthracyclines. / Prokineticins are angiogenic hormones that activate two G protein-coupled receptors (GPCRs): PKR1 and PKR2. PKR1 has emerged as a critical mediator of cardiovascular homeostasis and cardioprotection. The aim of this thesis project was to develop a first non-peptide PKR1 agonist stimulates cardioprotection and cardiac regeneration in mouse model of myocardial infarction (MI) or anti-cancer drug mediated cardiotoxicity. Collaboration with chemist and biomodelization team, we characterized the first selective/specific PKR1 agonist, named IS20. In vivo study demonstrated IS20 prevented cardiac lesion formation and improved cardiac function after myocardial infarction in mice, promoting proliferation of cardiac progenitor cells and neovasculogenesis (Gasser et al., 2015). Since use of a very potent anthracycline chemotherapeutic, Doxorubicin (DOX) is limited by cardiotoxicity, we hypothesized that IS20 could protect heart against DOX-mediated cardiotoxicity. Indeed, IS20 attenuated apoptosis induced by DOX in H9c2 cardiomyocytes and human epicardial progenitors in vitro. However, IS20 did not affect antineoplastic or cytostatic effect of DOX in cancer cell lines. In vivo, in the juvenile model of cardiotoxicity, IS20 significantly attenuated DOX-induced decrease in viability and proliferation cardiac progenitor cells. In the chronic cardiotoxicity model by DOX, IS20 improves heart structure and function by the activation of cardiac progenitor cells, diminishing cardiac cell death, improving vascular stability. IS20 has translational potential for cardioprotection in patients with cancer receiving anthracyclines.

The field of cancer research has grown immensely in recent decades and has led to a better understanding of the causes of the disease, as well as greatly improved treatment for various types of cancers, especially breast cancer. One of the most effective treatments involves the chemotherapeutic drug doxorubicin (DOX). DOX is an effective tool against all types of breast cancer, especially against triple negative breast cancer. However, DOX causes adverse side effects that include damage to the heart and skeletal muscle, particularly above specific cumulative doses. Recent evidence suggests that embryonic stem cell-derived (ES) exosomes, nanoscale extracellular vesicles that carry proteins, messenger RNA, and microRNAs, may be able to mitigate some of the cardio- and cytotoxic effects of DOX without reducing its efficacy. The present study examined the effects of combined treatment with DOX (1 μM) and ES exosomes (10 μg/mL) on three cancer cell lines, MCF7, MDA-MB-231, and MDA-MB-468. The DOX/ES exosomes treatment increased cell death and increased apoptosis specifically compared to control, as measured via dye exclusion assay and flow cytometry. The treatment also decreased cell growth compared to control, as measured via MTS cell proliferation assay. In addition, DOX/ES exosomes treatment also increased expression of pro-apoptotic Bax while decreasing the expression of anti-apoptotic Bcl-2, as measured via Western blot. Finally, the DOX/ES exosomes treatment decreased expression of miR-200c, a microRNA associated with preventing epithelial-mesenchymal transition, a process that is integral to metastasis. Although increased cell death and apoptosis and decreased cell proliferation implies that the DOX/exosomes treatment is effective against cancer, the decrease in miR-200c expression may suggest the opposite and will be investigated further in future studies. Even so, the results of this study suggest that exosomes may be an important component to reduce the harmful effects of cancer treatment in the future.

Cancer immunotherapy has proven to be challenging as it depends on overcoming multiple mechanisms that mediate immune tolerance to self-antigens. In situ immunization is based on the concept that it is possible to break immune tolerance by inducing tumor cell death in situ in a manner that provides antigen presenting cells such as dendritic cells (DCs) with a wide selection of tumor antigens that can then be presented to the immune system and result in a therapeutic anticancer immune response. Based on recent advances in the understanding of antitumor immunity, we designed a three-step approach to in situ immunization to lymphoma: (1) Inducing immunogenic tumor cell death with the chemotherapeutic drug Doxorubicin (Dox). Dox enhances the expression of "eat-me" signals by dying tumor cells, facilitating their phagocytosis by dendritic cells (DCs). Due to the vesicant activity of Dox, microparticles (MPs) made of PLGA (a biodegradable polymer) can safely deliver Dox intratumorally and are effective vaccine adjuvants; (2) Enhancing antigen presentation and T cell activation using anti-OX40; (3) Sustaining T cell responses by checkpoint blockade using anti-CTLA-4. In vitro, Dox MPs were less cytotoxic to DCs than to B lymphoma cells, did not require internalization by the lymphoma cells, and significantly enhanced phagocytosis of tumor cells by DCs as compared to soluble Dox. In mice, this three-step therapy induced CD4- and CD8-dependent systemic immune responses that enhanced T cell infiltration into distant lymphoma tumors leading to their eradication and significantly improving survival. Our findings demonstrate that systemic antitumor immune responses can be generated locally by three-step therapy and merit further investigation of three-step therapy for immunotherapy of lymphoma patients. Furthermore, we designed another in situ immunization approach using PLGA MPs loaded with both Dox and CpG oligodeoxynucleotides (CpG). The addition of CpG was to further enhance the Dox MP design by including an agent that addresses Step Two in situ, by enhancing tumor antigen presentation by DCs. In vitro, we show that Dox/CpG MPs can kill B and T lymphoma cells and are less toxic to DCs than soluble Dox. In vivo, Dox/CpG MPs combined with anti-CTLA-4 and anti-OX40 generated systemic immune responses that suppressed injected and distant tumors in a murine B lymphoma model, leading to tumor-free mice. The combination regimen was also effective at reducing T cell lymphoma and melanoma tumor burdens. In conclusion, Dox/CpG MPs represent a versatile, efficient and safe tool for in situ immunization that could provide a promising component of immunotherapy for patients with a variety of types of cancer.

Poly(acrylic acid) (PAA) was grafted onto both termini of Pluronic F87 (PEO₆₇-PPO₃₉-PEO₆₇) via atom transfer radical polymerization to produce a novel muco-adhesive block copolymer PAA₈₀-b-F₈₇-b-PAA₈₀. It was observed that PAA₈₀-F₈₇-PAA₈₀ forms stable complexes with weakly basic anti-cancer drug, Doxorubicin. Thermodynamic changes due to the drug binding to the copolymer were assessed at different pH by isothermal titration calorimetry (ITC). The formation of the polymer/drug complexes was studied by turbidimetric titration and dynamic light scattering. Doxorubicin and PAA-b-F87-b-PAA block copolymer are found to interact strongly in aqueous solution via non-covalent interactions over a wide pH range. At pH>4.35, drug binding is due to electrostatic interactions. Hydrogen-bond also plays a role in the stabilization of the PAA₈₀-F₈₇-PAA₈₀/DOX complex. At pH 7.4 (α=0.8), the size and stability of polymer/drug complex depend strongly on the doxorubicin concentration. When CDOX <0.13mM, the PAA₈₀-F₈₇-PAA₈₀ copolymer forms stable inter-chain complexes with DOX (110 ~ 150 nm). When CDOX >0.13mM, as suggested by the light scattering result, the reorganization of the polymer/drug complex is believed to occur. With further addition of DOX (CDOX >0.34mM), sharp increase in the turbidity indicates the formation of large aggregates, followed by phase separation. The onset of a sharp enthalpy increase corresponds to the formation of a stoichiometric complex. / Singapore-MIT Alliance (SMA)

In the recent years, development of various organic and inorganic nano-sized systems has gained great interests especially for cancer diagnosis and treatment and intense researches are carried out in this area. Regarding to the recent trends for drug delivery system design, the novel approaches for drug carriers are mainly based on development of smart and nano-size drug carriers which are targeted to cancer cells. Hence, for an effective tumor-targeted delivery device, besides its chemical structure further criteria such as detection of tumor site and sensitivity to the higher temperature and lower pH of the tumor compare to rest of the body gains importance. The aim of this study is to design and prepare polysebacic anhydride (PSA) based nanocapsules (NCs) loaded with Doxorubicin (DOX) which is an anti cancer drug. In order to obtain an intelligent delivery system, drug-loaded nanocapsules were coated with pH sensitive poly (L-histidine). PSA nano-carriers were firstly loaded with DOX and then in order to introduce pH sensitivity, they were coated with poly (L-histidine). PLH-coated NCs were modified with polyethylene glycol (PEG) to prevent their macrophage uptake. Drug release profile from this system was examined in two different buffer solutions prepared as acidic (pH 4) and physiological (pH 7.4) media. The physical and chemical properties of the nano particles were characterized by Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), ultraviolet and visible absorption spectroscopy (UV-VIS), and scanning electron microscopy (SEM). In vitro studies of the prepared nanocapsules were performed on MDA-MB-231 breast cancer cells by using WST Kit 8 cell viability test. In order to obtained results, pH sensitive nanocapsules with size 230 nm exhibited cellular uptake and promising intracellular release of drug.

With the recent advent of gene therapy, anti-apoptotic therapy has been receiving spotlight as a potential modality to inhibit the deterioration of pump function in the failing heart. We hypothesized that anti-apoptotic therapy using survivin gene delivery will 1) salvage H9c2 cells exposed to doxorubicin toxicity, and 2) ameliorate the progressive decline in left ventricular function in a rat model of doxorubicin-induced cardiomyopathy. The in vitro data suggested that survivin successfully prevented cell death under doxorubicin stress by both direct and indirect/paracrine mechanisms. Doxorubicin-treated animals developed progressive left ventricular dysfunction as evident by echocardiography and invasive pressure-volume loop analysis, which was prevented by ultrasound-mediated survivin plasmid delivery, but not empty plasmid delivery. Post-mortem analysis of myocardial tissue indicated a lowered apoptotic index in survivin-treated hearts, with evidence of decrease in interstitial fibrosis. In conclusion, survivin gene therapy was shown to ameliorate doxorubicin-induced cardiomyopathy, by decreasing apoptosis and preventing adverse remodeling.