Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Introduction: Doxorubicin (DOX), infamously known as the “red devil,” is considered the
most effective antineoplastic drug utilized in oncologic practice today. However, its clinical
use is hampered due to cumulative, dose-dependent cardiotoxicity, which can lead to reduced
quality of life, irreversible heart failure and death. The mechanisms involved in the
pathogenesis of cardiotoxicity have not been fully elucidated, but have previously been
demonstrated to involve oxidative stress, calcium dysregulation and mitochondrial
dysfunction. Since the mitochondria play a critical role in generation of reactive oxygen
species, the maintenance of calcium homeostasis and are the most extensively damaged by
DOX, they have become the main focus of novel therapeutic interventions. The morphology
and function of these dynamic organelles are regulated in part by mitochondrial fission and
fusion events, as well as mitochondrial quality control systems. Since mitochondrial
morphology is often associated with crucial cellular functions, this study aimed to investigate
the long-term effects of DOX on mitochondrial dynamics and the mitochondrial quality
control systems, mitophagy and the ubiquitin-proteasome pathway (UPP). Additionally, since
the mitochondria and the endoplasmic reticulum (ER) are two interconnected organelles, and
both play a role in maintaining calcium homeostasis, this study further assessed the effects of
chronic DOX treatment on ER function and calcium status.
Materials and Methods: In order to fully establish the effect of chronic DOX treatment in
vitro, two cardiac cell lines were utilized in this study. H9C2 cardiomyoblasts and humanderived
Girardi heart cells were cultured under standard culture conditions until ± 70-80%
confluency was reached, where after treatment commenced. Cells were treated daily with 0.2
and 1.0 μM of DOX for 96 and 120 hours in order to simulate chronic, cumulative
cardiotoxicity. Cell viability and apoptotic cell death were assessed with the MTT assay and
Caspase Glo 3/7 assays, respectively. The expression of proteins involved in mitochondrial
dynamics, mitochondrial biogenesis, the ubiquitin-proteasome pathway, mitophagy and ER
stress were determined with Western blotting. Organelle morphology was visualized with
fluorescence microscopy, and flow cytometry was used to assess mitochondrial and ER load.
In order to determine the oxidative capacity, stress and status within the cells following
treatment, the Oxygen radical absorbance capacity (ORAC), Thiobarbituric acid reactive
substances (TBARS) and Glutathione (GSH) assays were employed respectively. Finally,
intracellular and mitochondrial calcium was assessed and quantified with superresolution
structured illumination microscopy (SR-SIM) and flow cytometry respectively.
Results: DOX significantly reduced cell viability and increased apoptosis in both in vitro
cardiac cell models. This study further demonstrated that the expression of mitochondrial
fusion proteins, Mfn 1 and Mfn 2 were significantly downregulated, whilst the regulators of
fission, Drp1 and hFis1, were significantly elevated, therefore shifting the balance of
mitochondrial dynamics towards fission. Unopposed and elevated mitochondrial fission was
clearly evident from the morphology of these organelles, which displayed short, highly
fragmented mitochondria with a dispersed network following treatment. Chronic DOX also
downregulated the regulator of mitochondrial biogenesis, PGC-1α, thus inhibiting the
formation of new, functional mitochondria. The E3 ligases, MARCH5 and Parkin were
highly upregulated following treatment, indicating activation of the UPP and mitophagy.
Although chronic DOX stimulated K48 ubiquitination following treatment, it inhibited the
catalytic activity of the 26S proteasome, therefore blocking proteasomal degradation.
Although the antioxidant capacity (measured as ORAC) was significantly enhanced by both
concentrations of DOX, an increase in oxidative stress status was shown following DOX
treatment. In this regard lipid peroxidation significantly increased, while redox status of the
endogenous antioxidant, glutathione, significantly decreased. Additionally chronic DOX
treatment induced ER stress, which lead to an increase in cytosolic and mitochondrial
calcium. In response to ER stress, the unfolded protein response (UPR) was then stimulated.
Discussion: Results from this study indicate that chronic DOX treatment disrupts the
balance of mitochondrial dynamics, favouring mitochondrial fission. Mitochondrial
fragmentation is mediated by the downregulation of fusion proteins regulated by the E3
ubiquitin ligase, MARCH5 as well as by the increase in mitochondrial calcium.
Mitochondrial fission results in mitophagy, an adaptive response to protect the cardiac cell
against damaged mitochondria. This study also indicates that during chronic DOX-induced
cardiotoxicity ER stress and the UPR are induced, which is possibly responsible for the
disruption in calcium homeostasis. The inhibition of mitochondrial biogenesis coupled with
elevated mitophagy as observed in this chronic study, elucidates a plausible mechanism
whereby DOX induces mitochondrial dysfunction. Unregulated mitochondrial fragmentation
and inhibited mitochondrial biogenesis are known to regulate various cardiomyopathies, therefore since both these effects are induced by chronic DOX treatment suggests a
mechanism whereby cardiotoxicity, and ultimately heart failure are produced. This study
provides new insight into the role of chronic DOX plays in altering mitochondrial dynamics
and mitochondrial quality control systems. Further investigations targeted at limiting
mitochondrial fission may reduce the cardiovascular side effects associated with DOX. / AFRIKAANSE OPSOMMING: Inleiding: Doksorubisien (DOX), ook bekend as die “rooiduiwel,” word beskou as die mees
effektiewe anti-neoplastiese middel wat tans in onkologie praktyke gebruik word. Die
kliniese gebruik hiervan word gerem deur die kumulatiewe dosis-afhanklike kardiotoksisiteit
wat tot verlaagde lewenskwaliteit, onomkeerbare hartversaking, en tot die dood kan lei. Die
meganismes wat by die kardiotoksiese patogenese betrokke is, is nog onbekend, maar die
meganisme het moontlik te doen met oksidatiewe stres, kalsiumwanregulering en
mitochondriale wanfunksionering. Omrede die mitochondria ‘n kritieke rol in die vorming
van reaktiewe suurstofspesies speel, asook die handhawing van kalsiumhomeostase en die
mees beskadigde organelle deur DOX, het die hooffokus na nuwe terapeutiese intervensies
verskuif. Die morfologie en funskie van hierdie dinamiese organelle word gereguleer deels
deur mitochondriale fragmentering en fussie, asook mitochondriale kwaliteitsbeheersisteme.
Omrede mitochondriale morfologie geassosieer is met noodsaaklike sellulêre funksies, het
hierdie studie gepoog om die langtermyneffkte van DOX op mitochondriale dinamika en die
mitochondriale kwaliteitsbeheersisteme, mitofagie en die ubikwitien-proteosoomweg (UPW)
te ondersoek. Siende dat die mitochondria en die endoplasmiese retikulum (ER) twee
interverweefde organelle is, en beide ‘n rol speel in die handhawing van kalsiumhomeostase,
het hierdie studie verder die effekte van chroniese DOX behandeling op ER funksie en
kalsiumstatus ondersoek.
Materiaal en Metodes: Om die effek van chroniese DOX behandeling in vitro te verstaan in
hierdie studie, is twee hartsellyne gebruik. H9C2 kardiomioblaste en menslike Girardi
hartselle is onder standaardtoestande tot ± 70-80% konfluensie bereik is gekweek, waarna
behandeling begin is. Selle is daagliks met 0.2 en 1.0 μM DOX vir 96 en 120 uur behandel
om chroniese en kumulatiewe kardiotoksisiteit n ate boots. Selvatbaarheid en apoptotiese
seldood is onderskeidelik ondersoek deur middel van die MTT en Caspase Glo 3/7 toetse.
Die proteïenuitdrukking betrokke by mitochondriale dinamika, mitochondriale biogenese, die
ubikwitien-proteosoom weg, mitofagie en ER stres is deur middel van westerse afblatting
bepaal. Organelmorfologie is deur middel van fluoresensie mikroskopie gevisualiseer, en
vloeisitometrie was gebruik om die aantal mitochondria en ER lading te bepaal. Om die
oksidatiewe kapasiteit, stres en status binne die selle na behandeling te bepaal, is die ORAC,
TBARS en GSH toetse onderskeidelik gebruik. Laastens was die intrasellulêre en
mitochondriale kalsium ondersoek en gekwantifiseer met superresolussie gestruktureerde
illuminasie mikroskopie (SR-SIM) en vloeisitomerie.
Resultate: DOX het selvatbaarheid betekenisvol verlaag en apoptose in beide in vitro
kardiale selmodelle verhoog. Hierdie studie het verder aangetoon dat die uitdrukking van
mitochondriale fussie proteïene, Mfn 1 en Mfn 2 betekenisvol afgereguleer is, terwyl die
reguleerders van fragmentering, Drp1 en hFis1, betekenisvol verhoog is en daardeur die
balans van mitochondriale dinamika na fussie verskuif. Onverhinderde en verhoogde
mitochondriale fragmentering is duidelik sigbaar deur die morfologie van die organelle, wat
as kort, hoogsgefragmenteerde mitochondria met ‘n verspreide netwerk na behandeling
vertoon. Chroniese DOX het ook die mitochondriale biogenese reguleerder, PGC-1α,
afgereguleer en daardeur die vorming van nuwe, funksionele mitochondria geinhibeer. Die
E3 ligase, MARCH5 en Parkin is hoogs opgereguleer na behandeling, wat aktivering van
UPW en mitofagie aantoon. Alhoewel chroniese DOX K48 ubikwitinering na behandeling
gestimuleer het, het dit die katalitiese aktiwiteit van die 26S proteasoom geinhibeer en dus
die proteosomale degradasie geblokkeer. Antioksidantkapasiteit en oksidatiewe status was
betekenisvol na behandeling wat gevolglik tot hoë vlakke oksidatiewe skade binne die selle
gelei het. Addisioneel het chroniese DOX behandeling ER stres geïnduseer wat tot ‘n
toename in sitosoliese en mitochondriale kalsium gelei het. In reaksie op die ER stres is die
UPW gestimuleer.
Bespreking: Resultate van hierdie studie het aangetoon dat chroniese DOX behandeling die
balans van mitochondriale dinamika onderbreek en sodoende mitochondriale fragmentering
bevoordeel. Mitochondriale fragmentering word gemediëer deur die afregulering van fussie
proteïene wat deur die E3 ubikwitienligase, MARCH5, gereguleer word, en ook deur die
toename in mitochondriale kalsium. Mitochondriale fragmentering induseer mitofagie, ‘n
aanpassingsreaksie om die hartselle teen beskadigde mitochondria te beskerm. Hierdie studie
toon verder ook dat gedurende chroniese DOX-geïnduseerde ER stres, word die UPW ook
geïnduseer, wat moontlik dan verantwoordelik is vir die ontwrigting van kalsiumhomeostase.
Die inhibering van mitochondriale biogenese gekoppel met verhoogde mitofagie soos
waargeneem in hierdie studie, verklaar ‘n moontlike meganisme waardeur DOX
mitochondriale wanfunksionering veroorsaak. Ongereguleerde mitochondriale fragmentering
en geinhibeerde mitochondriale biogenese is bekend om verskeie kardiomiopatieë te reguleer.
Omrede beide hierdie effekte geinduseer word deur chroniese DOX behandeling kan dit
moontlik ‘n meganisme voorstel waarby kardiotokiese en uiteindelik hartversaking
ontwikkel. Hierdie studie bied nuwe insig in die rol wat chroniese DOX speel in die
wysiging van mitochondriale- dinamika en kwaliteitskontrole sisteme. Verdere ondersoeke
wat die mitochondriale fragmentering kan verminder mag moontlik die kardiovaskulêre
newe-effekte wat met DOX behandeling geassosieer is, verlaag. / National Research Foundation (NRF)
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/96676 |
| Date | 04 1900 |
| Creators | Opperman, Caleigh Margaret |
| Contributors | Sishi, Balindiwe J. N., Van Rooyen, Jacques, Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | xxii, 201 pages : illustrations |
| Rights | Stellenbosch University |
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