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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/20305 |
| Date | 03 1900 |
| Creators | Sishi, Balindiwe J. N. (Balindiwe Jennifer Nonkosazana) |
| Contributors | Engelbrecht, Anna-Mart, Loos, Benjamin, 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 | 216 p. : ill. |
| Rights | Stellenbosch University |
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