Lipotoxic cardiomyopathy increases the risk of heart failure in obese patients by adversely altering heart structure and function via toxic lipid specie mediated cellular stress and cell death. Increased fatty acid uptake and esterification in cardiomyocytes increases toxic lipid intermediates. These cardiotoxic lipid species such as diacylglycerol have recently been shown to deacidify lysosomes in cardiomyocytes by activating protein kinase C βII mediated NADPH oxidase 2 generation of superoxide that inhibits proton pumps on lysosomal membranes by S-nitrosylation. Autophagy, a lysosome dependent cellular survival process, is impaired upon cardiomyocyte lipid-overload due to inhibition of pH-dependent proteolytic autophagosome degradation in the lysosome. Subsequent accumulation of autophagic vesicles heightens cardiomyocyte sensitization to additional stresses of ischemia-reperfusion or ER dysfunction, culminating in impaired cardiac metabolic flexibility leading to cell death. Low cardiomyocyte regenerative capacity calls for strategies to preserve cell number in states of increased stress, such as lipid-induced impairment of autophagy. Lysosome-targeted reacidifying devices can provide an effective means to restore autophagic flux. In this thesis, a therapeutic strategy utilizing poly(DL-lactide-co-glycolide) (PLGA) nanoparticle degradation to reacidify lysosomes and revert cardiotoxic lipid specie induced blockade in autophagic flux in cardiomyocytes is presented. Endocytosed PLGA acidic nanoparticles were designed to rapidly degrade and release acidic monomers in lysosomes to restore pH dependent phosphatase and cathepsin L activity in cardiomyocytes with acute lipotoxicity. Optimized pre-palmitate treatment periods demonstrated that PLGA nanoparticles with polyethylenimine cationic surface coatings provide an effective restoration of autophagic flux in the presence of lipid-overload modeled by acute palmitate treatment in cardiomyocytes.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Introduction Cisplatin has been widely used to treat solid tumours and much success has come from the use of this drug in the treatment of head and neck, ovarian, testicular, cervical and small-cell lung cancers. However, the success of cisplatin treatment is limited due to its dose-limiting toxicity and its resulting side-effects, such as nephro- and ototoxicity. The devastating side-effects induced by cisplatin treatment provided the platform for this study whereby the aim was to lower the concentration of cisplatin while maintaining its cancer-specific cytotoxic action. Equally concerning is, cisplatin resistance which is becoming increasingly common, and this radically limits the clinical efficacy and utility of the drug. Adjuvant therapy has thus become necessary in an attempt to possibly curb or lessen the extent of cisplatin resistance. Due to the large body of evidence implicating the importance of autophagy in cancer, the prospect of targeting this mechanism has generally been accepted. Various chemotherapy agents induce autophagy in cancer cells; however the effect of cisplatin on autophagic induction has not been very well explored. We thus hypothesise that the manipulation of the autophagic pathway will sensitise cancer cells to a low concentration of cisplatin treatment. Furthermore, due to the functional interaction between Bcl-2 and Beclin-1 and its role in the regulation of autophagy, ratio analysis of Beclin-1 to Bcl-2 as means of detecting the role of autophagy within the cell under homeostatic and treatment/stress conditions has been conducted. Additionally, Bcl-2 has a prominent role in the malignant cell and it’s over-expression has been found to confer resistance in a variety of cancerous cell lines. We therefore hypothesise that the silencing of Bcl-2 prior to cisplatin treatment will sensitise cervical cancer cells to apoptosis and increase the Beclin-1/Bcl-2 ratio in favour of apoptosis. Materials and Methods Three human cervical cell lines were used: a non-cancerous ectocervical epithelial cell line (Ect1/E6E7) and two cancerous cervical cell lines (HeLa and CaSki). In order to determine a concentration of cisplatin that was non-toxic to the non-cancerous Ect1/E6E7 cell line, a dose-response was performed. With the use of an autophagy inhibitor (bafilomycin A1) and an autophagy inducer (rapamycin), autophagic flux capacities were assessed in each cell line through the Western blotting technique. In order to assess whether the chosen concentration of cisplatin induced autophagy, flow cytometry with the use of a Lysotracker™ dye was utilised, as well as analysis of autophagy protein levels (LC-3 II, Beclin-1 and p62). Autophagy modulation was achieved through two methods: pharmacological modulation with use of two recognised agents, namely bafilomycin A1 and rapamycin, and biological manipulation with the use of ATG5 and mTOR mRNA silencing. The effects of different treatment regimes on cell death was assessed with the use of PARP and caspase-3 cleavage through Western blotting, caspase-3/-7 activity (Caspase-Glo®), PI inclusion, LDH release and MTT reductive capacity. Additionally the effects of these treatment regimes on cell-cycle progression were also analysed. Beclin-1 and Bcl-2 expression was determined through Western blotting and immunocytochemistry before and after treatment with cisplatin in HeLa and CaSki cells. To assess the reliance of the cervical cancer cells on Bcl-2 after cisplatin treatment, Bcl-2 knock-down was achieved through RNA interference, where after the Beclin-1/Bcl-2 ratio was assessed as well as apoptosis with the use of cleaved PARP analysis (Western blotting) and Caspase-Glo©. For the ex vivo analysis, biopsies were collected from patients undergoing routine colposcopy screenings and hysterectomies at Tygerberg Hospital, Tygerberg, Western Cape. A total of 10 normal, 29 low-grade squamous intraepithelial lesions (LSIL), 33 high-grade squamous intraepithelial lesions (HSIL) and 13 carcinoma biopsies were collected for analysis, where after the expression profiles of two autophagy markers (mTOR and LC-3 II), as well as one anti-apoptotic marker (Bcl-2) were assessed. Protein levels were analysed through Western blot and confirmed through immunohistochemistry. Results Dose-response curves revealed that 15 μM of cisplatin did not induce cell death in the normal cervical epithelial cell line (Ect1/E6E7) and was therefore utilised through-out the remainder of the study. It was additionally determined that the CaSki cells were more resistant to cisplatin treatment when compared to the HeLa and Ect1/E6E7 cells. Autophagic flux analysis revealed that, although all three cell lines were cervix derived, their autophagic flux capacities differed. It was observed that the chosen concentration of cisplatin was able to induce autophagy in all three cell lines, with the HeLa cells demonstrating a particularly pronounced response. Autophagy modulation in conjunction with cisplatin treatment revealed the following: Autophagy inhibition with bafilomycin A1 lead to significant increases in caspase-3 and PARP cleavage and LDH release in both cervical cancer cell lines. The inhibition of autophagy through silencing of ATG5 induced caspase-3 cleavage and agrees with results obtained from pharmacological inhibition of autophagy with bafilomycin A1. In addition to autophagic induction, a low concentration of cisplatin induced the up-regulation of Bcl-2, which when silenced significantly improved cisplatin-induced apoptosis in both cervical cancer cell lines. Analysis of the expression profiles of mTOR and LC-3 in normal, pre-malignant (LSIL and HSIL) and cancerous cervical tissue revealed that autophagy is significantly up-regulated in HSILs and carcinoma of the cervix. Additionally, Bcl-2 expression is significantly increased in cervical carcinoma tissue, which agrees with results from other studies. Conclusion Autophagic flux capacities between the three cell lines investigated, derived from the same organ, differ significantly. This should be taken into consideration when autophagic modulation is being used as an adjuvant treatment. With regard to chemotherapy treatment in cervical cells, a low-concentration of cisplatin significantly induces autophagy in malignant and non-malignant cervix-derived cell lines where it serves a pro-survival mechanism. Inhibition of autophagy with bafilomycin A1 and ATG5 siRNA confirmed this survival effect in both cancerous cell lines where apoptosis was significantly increased. Interestingly, rapamycin pre-treatment together with cisplatin did not induce significant levels of apoptosis in HeLa cells where autophagy induction may have provided additional protection from the cytotoxic effects of cisplatin. Therefore the inhibition of autophagy through pharmacological and biological inhibition improves the cytotoxicity of a low concentration of cisplatin and provides a promising new avenue for the future treatment of cervical cancer. Bcl-2 up-regulation in response to cisplatin treatment also serves as a protective mechanism by which cervical cancer cells survive. The extent of apoptotic cell death observed after biological inhibition of Bcl-2 reiterates the fact that this response may be exploited in order to favour the use of lower concentrations of cisplatin. Analysis of clinical specimens emphasised the value of the in vitro work: Cervical cancer biopsies had increased expression of both LC-3 II and Bcl-2, indicating autophagy induction and apoptosis inhibition, respectively. Thus two novel methods of improving cisplatin cytotoxicity have been demonstrated in the following study. Treatment regimens may administer more frequently and prolonged due to the minimal side-effects that accompanies low-dose cisplatin treatment. / AFRIKAANSE OPSOMMING: Inleiding Sisplatien word algemeen gebruik vir die behandeling van soliede gewasse. Baie sukses is reeds deur die gebruik van díe middel behaal in die behandeling van kop en nek, ovariale, terstikulêre, servikale en klein-sel kankers. Die sukses van Sisplatien-behandeling word wel ingeperk deur die dosis-beperkende toksisiteit en die gevolglike newe-effekte soos nefrotoksisiteit. Hierdie verwoestende newe-effekte wat deur sisplatien behandelings geïnduseer word, het as die platform vir hierdie studie gedien. Die doel was om die sisplatien konsentrasies te verlaag, maar terselfdertyd die kankerspesifieke sitotoksisiteit te behou. Nog ʼn punt van kommer is dat sisplatien-weerstandigheid aan die toeneem is, wat die kliniese effektiwiteit en gebruik van hierdie middel geweldig beperk. Byvoegmiddels het dus noodsaaklik geraak in die poging om die sisplatien-weerstandigheid te verhoed. As gevolg van verskeie bewyse wat die belangrikheid van outofagie in kanker impliseer, is die vooruitsig om hierdie meganisme te teiken, algemeen aanvaar. Verskeie chemoterapeutiese middels induseer outofagie in kanker selle, hoewel die effek van Sisplatien op outofagiese induksie nog nie goed ondersoek is nie. Ons hipotese is dus dat die manipulasie van die outofagiese pad die kankerselle sensitiseer tot ʼn lae konsentrasie van sisplatien. Verder, as gevolg van die funksionele interaksie tussen Bcl-2 en Beclin-1, en hul rol in die regulering van outofagie, is verhouding-analises van Beclin-1 tot Bcl-2 uitgevoer met die doel om die rol van outofagie in die sel onder homeostatiese en behandeling/stres kondisies te bepaal. Verder is Bcl-2 bekend daarvoor om ʼn prominente rol te speel in kwaadaardige selle, en die ooruitdrukking daarvan is gevind om weerstandigheid aan te help in ʼn verskeidenheid van kankeragtige sellyne. Ons hipotetiseer dus dat geenonderdrukking van Bcl-2 voor die behandeling met sisplatien die servikale kanker selle sal sensitiseer tot apoptose en ʼn verhoging in die verhouding van Beclin-1/Bcl-2 veroorsaak, wat in die guns van apoptose is. Materiale en Metodes Drie menslike servikale sellyne was gebruik: ʼn nie-kankeragtige servikale epiteel sellyn (Ect/E6E7) en twee kankeragtige servikale sellyne (HeLa en CaSki). Om ʼn konsentrasie van sisplatien te bepaal wat nie-toksies tot die nie-kankeragtige Ect1/E6E7 sellyn is, was ʼn dosisrespons uitgevoer. Met die gebruik van ʼn outofagiese inhibeerder (bafilomycin A1) en ʼn outofagiese induseerder (rapamycin), is die outofagiese-fluks kapasiteite van elke sellyn deur die Western Blotting tegniek geassesseer. Om te bepaal of die gekose konsentrasie van sisplatien outofagie induseer, is vloeisitometrie met ʼn Lysotracker™ kleurstof gebruik, sowel as analises op outofagie proteïenvlakke (LC-3 II, Beclin-1 en p62). Outofagie modulering is behaal deur twee metodes: farmakologiese modulering met twee erkende middels, naamlik bafilomycin A1 en rapamycin, en biologiese manipulasie met die gebruik van ATG5 en mTOR geenonderdrukking. Die effekte van die verskillende behandeling skedules op seldood was geassesseer deur gebruik te maak van PARP en kaspase-3 splitsing deur Western Blotting, kaspase-3/-7 aktiwiteit deur Caspase-Glo ®, PI-insluiting, LDH vrystelling en MTT reduserende kapasiteit. Verder is die effekte van hierdie behandeling skedules op selsiklus progressie ook geanaliseer. Beclin-1 en Bcl-2 uitdrukking was ook bepaal deur Western Blotting en immunohistochemie voor en na behandeling met sisplatien in HeLa en CaSki selle. Om die afhanklikheid van die servikale kankerselle op Bcl-2 na sisplatien behandelings te toets, is Bcl-2 onderdruk deur RNA-inmenging, waarna Beclin-1/Bcl-2 verhouding geassesseer is, sowel as opoptose deur die gebruik van gesplitste PARP analises (Western Blotting) en Caspase-Glo©. Vir die ex vivo analises is biopsies vanaf pasiënte wat roetine kolposkopie en histerektomies ondergaan, verkry (Tygerberg Hospitaal, Tygerberg, Westelike Provinsie). ʼn Totaal van 10 normale, 29 lae-graad plaveisel intraepiteel letsels (LSIL), 33 hoe-graad plaveisel intraepiteel letsels (HSIL) en 13 karsinoom biopsies is verkry vir analises. Die uitdrukkingsprofiel van twee outofagiese merkers (mTOR en LC-3 II), asook een merker vir apoptose (Bcl-2), was geassesseer. Proteïen vlakke was ook deur Western Blotting geanaliseer en deur immunohistochemie bevestig. Resultate Dosisrespons kurwes het getoon dat 15 μM sisplatien nie seldood in die normale sellyn (Ect1/E6E7) geïnduseer het nie, en was daarom gebruik deur die res van hierdie studie. Verder is daar ook gevind dat CaSki selle meer weerstandig tot sisplatien behandelings is wanneer vergelyk word met die HeLa en Ect1/E6E7 selle. Outofagiese-fluks analises het getoon dat, alhoewel al drie sellyne vanaf die serviks afkomstig is, daar verskille is in hul outofagiese-fluks kapasiteit. Daar is ook waargeneem dat die gekose konsentrasie van sisplatien in staat was om outofagie te induseer in al drie sellyne, met HeLa selle wat die mees merkbare respons getoon het. Modulering van outofagie in samewerking met sisplatien behandelings het die volgende onthul: inhibisie van outofagie deur bafilomycin A1 het gelei tot ʼn beduidende verhoging in kaspase-3, PARP splitsing en LDH vrylating in beide servikale kankersellyne. Geenonderdrukking van ATG5 induseer kaspase-3 splitsing en stem ooreen met resultate wat verkry is deur farmakologiese inhibisie van outofagie met bafilomycin A1. Bykomend tot outofagiese indusering, het ʼn lae konsentrasie sisplatien die opregulering van Bcl-2 geïnduseer. Wanneer Bcl-2 geenonderdrukking in hierdie scenario toegepas was, het dit ʼn beduidende verbetering in sisplatien-geïnduseerde apoptose in beide servikale kankersellyne getoon. Analises van die uitdrukkingsprofiel van mTOR en LC-3 in normale, pre-maligne (LSIL en HSIL) en kankeragtige servikale weefsel, het getoon dat outofagie beduidend opgereguleer is in HSILs en servikale karsinome. Verder is Bcl-2 uitdrukking ook gevind om beduidend verhoog te wees in servikale karsinoomweefsel, wat ooreenstem met resultate verkry in ander studies. Gevolgtrekking Outofagiese-fluks kapasiteite tussen die drie sellyne, afkomstig van dieselfde orgaan, toon beduidende verskille. Hierdie bevinding moet in ag geneem word wanneer outofagiese-modulering as ʼn bevorderingsbehandeling gebruik word. Met betrekking tot chemoterapie behandeling in servikale selle; ʼn lae konsentrasie van sisplatien veroorsaak ʼn beduidende indusering van outofagie in kwaadaardige en nie-kwaadaardige serviks-afkomstige sellyne, en dien as ʼn oorlewingsmeganisme. Inhibisie van outofagie met bafilomycin A1 en ATG5 siRNA het hierdie beskermings effek bevestig, aangesien apoptose beduidend verhoog was in beide kankersellyne. Interessant genoeg het rapamycin pre-behandeling tesame met sisplatien nie beduidende vlakke van apoptose in HeLa selle geïnduseer nie. Outofagie induksie mag dalk addisionele beskerming teen die sitotoksiese effekte van sisplatien gebied het. Daarom het die inhibisie van outofagie deur farmakologiese en biologiese inhibering die sitotoksisiteit van ʼn lae konsentrasie sisplatien bevorder, wat ʼn belowende bevinding is vir die toekomstige behandeling van servikale kanker. Bcl-2 opregulering as gevolg van sisplatien behandelings dien ook as beskermings meganisme waarby servikale kankerselle oorleef. Die mate van apoptotiese seldood wat waargeneem word na biologiese inhibering van Bcl-2, wys weer op die feit dat hierdie respons uitgebuit kan word vir die gebruik van laer konsentrasies van sisplatien. Analises van die kliniese monsters het ook die waarde van die in vitro werk versterk: Servikale kanker biopsies het verhoogde uitdrukking van beide LC-3 II en Bcl-2 getoon, wat aandui dat outofagie geïnduseer en apoptose geïnhibeer word. Daar is dus twee nuwe metodes vir die verbetering van sisplatien-toksisiteit in hierdie studie gedemonstreer. Behandeling regimes kan meer gereeld en vir langer tydperke toegepas word, aangesien die newe-effekte van lae-dosis sisplatien behandelings minimaal is. / MRC for funding

Très peu de données sont aujourd’hui disponibles sur le rôle des nutriments et de leurs interactions in vivo dans la régulation de l’autophagie. L'objectif principal de cette thèse était donc d’étudier l’effet de la qualité nutritionnelle de l’aliment sur le contrôle de cette fonction cellulaire. Une première étude a permis de montrer que les différents ratios en protéines et en glucides influencent significativement les mécanismes de contrôle de l’autophagie dans le muscle de truite arc-en-ciel (Oncorhynchus mykiss). Ces résultats ont été renforcés par une étude sur culture primaire de myoblaste de truite montrant que l’addition d’acides aminés dans le milieu de culture inhibe l’autophagie alors que le glucose à un effet inverse (article 1). Une seconde étude a porté sur la fraction protéique de l’aliment et notamment sur la teneur en méthionine, dont le niveau est trop faible dans les aliments pour poissons à base de végétaux. Les résultats obtenus indiquent que la machinerie autophagosomale mais également les principaux facteurs du renouvellement des protéines musculaires sont sensible aux variations de la teneur en méthionine de l’aliment et que la réponse qui en résulte peut fortement affecter la croissance (article 2). L’ensemble des données obtenues dans les deux premières études reposaient sur la mesure du taux d’un marqueur de l’autophagie (LC3-II) qui est à la fois produite et dégradée au cours du processus (flux) autophagique. Ainsi, dans l’optique de préciser les résultats obtenus dans les deux premiers articles, une troisième étude a été effectuée afin de déterminer s’il est possible de bloquer le flux autophagique dans le muscle de truite par l’emploi de différents agents pharmacologiques (inhibiteurs du flux autophagique). Il s’agissait également de déterminer les limites de l’utilisation de tels inhibiteurs chez cette espèce. Les essais effectués n’ont pas permis de mesurer le flux autophagique dans le muscle. En revanche, l’injection intrapéritonéale de colchicine a bien bloqué le flux autophagique dans le foie, ouvrant ainsi un nouveau champ d’investigations sur le rôle de l’autophagie dans le métabolisme intermédiaire. En conclusion, l’ensemble de ces travaux montre que l’autophagie n’est pas uniquement sensible à l’état nutritionnel (jeûne/nourris) mais également à la nature des aliments consommés. Outre leurs intérêts agronomique et thérapeutique, ces résultats ouvrent de nouvelles perspectives pour une meilleure compréhension des mécanismes d’action de l’autophagie au niveau cellulaire et métabolique mais également de son rôle dans l’adaptation des espèces au cours l’évolution. / Few data has been published on the role of nutrients and their interactions in vivo in the regulation of autophagy. The main objective of this thesis was therefore to characterize the response of the autophagic/lysosomal pathway to the macronutrients composition of the diets. The first study showed that different ratio of proteins and carbohydrates in the diet significantly affect the controls of autophagy in the muscle of rainbow trout (Oncorhynchus mykiss). These results were strengthened by study on primary culture of trout myoblasts showing that the addition of amino acids in cell culture medium inhibited the formation of autophagosomes, whereas the addition of glucose had an opposite effect (paper 1). A second study focused on the protein fraction of the diet and specifically on the content of methionine, whose the levels are low in fish feed plant-based diets. The obtained results showed that both autophagy machinery and the main factors of muscle protein turnover are significantly sensitive to change in dietary methionine levels and the resulting response may strongly affect growth and feed utilization (paper 2). The data obtained in these two first studies were based on measuring the level of autophagy marker (LC3-II), which is both produced and degraded during autophagic (flux) process. Thus, in view to clarify the results obtained in the two first studies, we conducted a third study to determine whether it is possible to block the autophagic flux in trout muscle by using different lysosomotropic agents (autophagic flux inhibitors). The objective was also to determine the limits of autophagic flux inhibitors utilisation in vivo. Different tests failed to measure autophagic flux in the muscle of rainbow trout. In contrast, intraperitoneal injection of colchicine blocked the autophagic flux in the liver. This study allowed us to investigate the function of autophagy in the intermediary metabolism. In conclusion, these studies show that autophagy is not only sensitive to the nutritional status (fasting/fed) but also to the nature of the consumed diets. In addition to their therapeutic and agricultural interests, these results open new perspectives to better understand the mechanisms of autophagy at metabolic and cellular level but also its role in the adaptation of species during evolution.