6 research outputs found
An investigation into the P13-K/AKT signalling pathway in TNF-a-induced muscle proeolysis in L6 myotubes
Thesis (MSc (Physiological Sciences))--Stellenbosch University, 2008.Introduction: Skeletal muscle atrophy is a mitigating complication that is characterized by
a reduction in muscle fibre cross-sectional area as well as protein content, reduced force,
elevated fatigability and insulin resistance. It seems to be a highly ordered and regulated
process and signs of this condition are often seen in inflammatory conditions such as cancer,
AIDS, diabetes and chronic heart failure (CHF). It has long been understood that an
imbalance between protein degradation (increase) and protein synthesis (decrease) both
contribute to the overall loss of muscle protein. Although the triggers that cause atrophy are
different, the loss of muscle mass in each case involves a common phenomenon that induces
muscle proteolysis. It is becoming evident that interactions among known proteolytic systems
(ubiquitin-proteosome) are actively involved in muscle proteolysis during atrophy. Factors
such as TNF-α and ROS are elevated in a wide variety of chronic inflammatory diseases in
which skeletal muscle proteolysis presents a lethal threat. There is an increasing body of
evidence that implies TNF-α may play a critical role in skeletal muscle atrophy in a number of
clinical settings but the mechanisms mediating its effects are not completely understood. It is
also now apparent that the transcription factor NF-κB is a key intracellular signal transducer
in muscle catabolic conditions. This study investigated the various proposed signalling
pathways that are modulated by increasing levels of TNF-α in a skeletal muscle cell line, in
order to synthesize our current understanding of the molecular regulation of muscle atrophy.
Materials and Methods: L6 (rat skeletal muscle) cells were cultured under standard
conditions where after reaching ± 60-65% confluency levels, differentiation was induced for a
maximum of 8 days. During the last 2 days, myotubes were incubated with increasing
concentrations of recombinant TNF-α (1, 3, 6 and 10 ng/ml) for a period of 40 minutes, 24
and 48 hours. The effects of TNF-α on proliferation and cell viability were measured by MTT
assay and Trypan Blue exclusion technique. Morphological assessment of cell death was
conducted using the Hoechst 33342 and Propidium Iodide staining method. Detection of
apoptosis was assessed by DNA isolation and fragmentation assay. The HE stain was used for
the measurement of cell size. In order to determine the source and amount of ROS production,
MitoTracker Red CM-H2 X ROS was utilised. Ubiquitin expression was assessed by
immunohistochemistry. PI3-K activity was calculated by using an ELISA assay and the
expression of signalling proteins was analysed by Western Blotting using phospho-specific and total antibodies. Additionally, the antioxidant Oxiprovin was used to investigate the
quantity of ROS production in TNF-α-induced muscle atrophy.
Results and Discussion: Incubation of L6 myotubes with increasing concentrations of
recombinant TNF-α revealed that the lower concentrations of TNF-α used were not toxic to
the cells but data analysis of cell death showed that 10 ng/ml TNF-α induced apoptosis and
necrosis. Long-term treatment with TNF-α resulted in an increase in the upregulation of TNF-
α receptors, specifically TNF-R1. The transcription factors NF-κB and FKHR were rapidly
activated thus resulting in the induction of the ubiquitin-proteosome pathway. Activation of
this pathway produced significant increases in the expression of E3 ubiquitin ligases MuRF-1
and MAFbx. Muscle fibre diameter appeared to have decreased with increasing TNF-α
concentrations in part due to the suppressed activity of the PI3-K/Akt pathway as well as
significant reductions in differentiation markers. Western blot analysis also showed that
certain MAPKs are activated in response to TNF-α. No profound changes were observed with
ROS production. Finally, the use Oxiprovin significantly lowered cell viability and ROS
production. These findings suggest that TNF-α may elicit strong catabolic effects on L6
myotubes in a dose and time dependent manner.
Conclusion: These observations suggest that TNF-α might have beneficial effects in
skeletal muscle in certain circumstances. This beneficial effect however is limited by several
aspects which include the concentration of TNF-α, cell type, time of exposure, culture
conditions, state of the cell (disturbed or normal) and the cells stage of differentiation. The
effect of TNF-α can be positive or negative depending on the concentration and time points
analysed. This action is mediated by various signal transduction pathways that are thought to
cooperate with each other. More understanding of these pathways as well as their subsequent
upstream and downstream constituents is obligatory to clarify the central mechanism/s that
control physiological and pathophysiological effects of TNF-α in skeletal muscle
Anthracycline-induced cardiotoxicity : the role of proteolytic pathways
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
Amino acid starvation sensitizes resistant breast cancer to doxorubicin-induced cell death
CITATION: Thomas, M., et al. 2020. Amino acid starvation sensitizes resistant breast cancer to doxorubicin-induced cell death. Frontiers in Cell and Developmental Biology, 8:565915,
doi:10.3389/fcell.2020.565915.The original publication is available at https://www.frontiersin.org/articles/10.3389/fcell.2020.565915/fullPublication of this article was funded by the Stellenbosch University Open Access FundMany clinical trials are beginning to assess the effectiveness of compounds known to regulate autophagy in patients receiving anti-cancer chemotherapy. However, autophagy inhibition, through exogenous inhibitors, or activation, through starvation, has revealed conflicting roles in cancer management and chemotherapeutic outcome. This study aimed to assess the effect of amino acid starvation on doxorubicin-treated breast cancer cells by assessing the roles of autophagy and apoptosis. An in vitro breast cancer model consisting of the normal breast epithelial MCF12A and the metastatic breast cancer MDAMB231 cells was used. Apoptotic and autophagic parameters were assessed following doxorubicin treatments, alone or in combination with bafilomycin, ATG5 siRNA or amino acid starvation. Inhibition of autophagy, through ATG5 siRNA or bafilomycin treatment, increased caspase activity and intracellular doxorubicin concentrations in MCF12A and MDAMB231 cells during doxorubicin treatment. While amino acid starvation increased autophagic activity and decreased caspase activity and intracellular doxorubicin concentrations in MCF12A cells, no changes in autophagic parameters or caspase activity were observed in MDAMB231 cells. Our in vivo data showed that 24 h protein starvation during high dose doxorubicin treatment resulted in increased survival of tumor-bearing GFP-LC3 mice. Results from this study suggest that short term starvation during doxorubicin chemotherapy may be a realistic avenue for adjuvant therapy, especially with regards to the protection of non-cancerous cells. More research is however, needed to fully understand the regulation of autophagic flux during starvation.https://www.frontiersin.org/articles/10.3389/fcell.2020.565915/fullPublisher's versio