Does cardioprotection by autophagy go beyond acute cardiotoxicity?

Chabaesele, Itumeleng (2016-03)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Introduction and Aim The discovery of Doxorubicin (DOX) in the 1960s has drastically improved the survival rates of cancer patients; however, its success is limited by dose-dependent cardiotoxicity. While much of the literature has focused on acute cardiotoxicity which is generally reversible, chronic cardiotoxicity is irreversible and poses a serious threat since it can lead to congestive heart failure. The mechanisms that contribute to cardiotoxicity are still a matter of controversy; however, oxidative stress-induced myocardial damage and apoptosis are thought to be the major role players. One of the best understood and most widely studied processes is autophagy, an evolutionary conserved pathway of intracellular degradation. Although it has been attributed to various cardiac disorders, an increasing body of evidence corroborates the notion that autophagy may serve as a probable therapeutic target by providing cardioprotection in different contexts. Materials Autophagy was induced in H9c2 cardiomyoblasts by rapamycin treatment, starvation and siRNA (mTOR). This process was also inhibited by bafilomycin A1 treatment. Chronic DOX cytotoxicity was induced by treating cells daily with 0.2 μM DOX for 120 hrs. In an effort to determine whether autophagy upregulation or downregulation was beneficial, cell viability, apoptosis, oxidative stress and mitochondrial membrane function were assessed by utilizing various assays, western blotting, fluorescence microscopy as well as flow cytometry. Results and Conclusion Autophagy stimulation via siRNA (mTOR) and autophagy inhibition with bafilomycin did not decrease the detrimental effects associated with long-term DOX cytotoxicity. These effects were instead aggravated and became progressively worse. However, autophagy upregulation through rapamycin or starvation proved beneficial in this context as the amount of DOX that normally accumulates in the cells was reduced, and mitochondrial function and damage substantially improved. These observations also lead to improved cell survival. Therefore, based on the above results, autophagy induction in this context may be used as a plausible adjuvant treatment strategy for the mitigation of DOX-induced cardiac damage.

AFRIKAANSE OPSOMMING: Inleiding en Doelwit Die ontdekking van Doksorubisien (DOX) in die 1960s het oorlewingstempo van kankerpasiënte drasties verbeter; alhoewel die sukses hieraan beperk is tot ‘n dosis afhanklike kardiotoksisiteit. Menige literatuur fokus op akute kardiotiksisiteit wat oor die algemeen omkeerbaar is, terwyl chroniese kardiotiksisiteit onomkeerbaar is wat ‘n groot risiko inhou en kan tot die ontstaan van kongestiewe hartversaking lei. Die bydraende kardiotoksisiteitmeganismes is steeds kontroversiëel, maar oksidatiewe stres-geïnduseerde miokardiale skade en apoptose word as moontlike hoofrolspelers ondersoek. Een van die mees begrypte, en bestudeerde proses is outofagie, ‘n evolusionêr gekonserveerde intra-sellulêre degradasie padweg. Alhoewel dit bydraend is tot verskeie kardiale afwykings het ‘n toenemende hoeveelheid bewyse aangetoon dat outofagie moontlik as ‘n terapeutiese teiken in kardiale beskerming in verskeie kontekse kan dien. Materiale Outofagie is in H9c2 kardiomioblaste deur rapamisienbehandeling, nutrientweerhouding, en siRNA (mTOR) geïnduseer. Hierdie proses is ook geinhibeer deur bafilomisien A1 behandeling. Chroniese DOX sitotoksisiteit is verkry deur die selle daagliks met 0.2 μM DOX vir 120 uur te behandel. In ‘n poging om vas te stel of outofagie opregulering of afregulering voordelig was, is selvatbaarheid, apoptose, oksidatiewe stres en mitochondriale membraanfunksionering deur verskeie toetse, “western blotting”, fluoresensie mikroskopie sowel as vloeisitometrie ondersoek. Resultate en Gevolgtrekking Outofagie stimulasie via siRNA (mTOR) en outofagie inhibering met bafilomisien het nie die skadelike effekte wat met langtermyn DOX sitotoksisiteit gepaard gaan verlaag nie. Inteendeel het hierdie effekte toenemend vererger. Outofagie opregulering deur rapamisien of nutrientweerhouding was voordelig in hierdie konteks, omrede die hoeveelheide DOX wat normaalweg in die selle ophoop, verlaag was en die mitochondriale funksionering en skade betekenisvol verbeter het. Hierdie waarnemings het tot verbeterde seloorlewing aanleiding gegee. Die resultate van hierdie studie toon dat outofagie induksie in hierdie konteks moontlik gebruik kan word as ‘n moontlike adjuvante behandelingstrategie vir die verligting van DOX-geïnduseerde kardiale beskadiging.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/98577
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