The (un)SAFE and RISK(y) sides of doxorubicin-induced cardiotoxicity

Goldswain, Toni Leigh (2014-12)

Thesis (MSc)--Stellenbosch University, 2014.

Thesis

ENGLISH ABSTRACT: Introduction The discovery of Doxorubicin 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 minor and generally reversible, chronic cardiotoxicity poses a serious threat to cancer survivors since it can lead to dilative cardiomyopathy, congestive heart failure and even death. 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. Reperfusion injury, also characterized by oxidative stress and apoptosis, occurs as a result of restoring blood flow to an ischemic heart. Fortunately, pre- and post-conditioning are techniques employed to minimize this damage and are thought to do so by activating the reperfusion injury salvage kinase (RISK) and survivor activating factor enhancement (SAFE) pathways. The RISK pathway involves the pro-survival kinases, Erk1/2 and Akt, while the SAFE pathway, triggered by TNF-α, involves Jak2 and STAT3. Since both reperfusion injury and Doxorubicin-induced cardiotoxicity share similar characteristics, this study aimed to determine whether the RISK and SAFE pathways are activated in response to long-term Doxorubicin treatment. Furthermore, this study aimed to determine whether TNF-α is produced during treatment, since its role in Doxorubicin-induced cardiotoxicity is still relatively unknown. Methods H9c2 cardiomyocytes and differentiated C2C12 myotubes were treated daily with increasing concentrations of Doxorubicin for a total of 120 hours. Cell viability, apoptosis and necrosis were assessed using the MTT, Caspase-Glo® 3/7 and lactate dehydrogenase assays respectively. TNF-α production was measured using Quantikine® ELISA kits and various assays were used to assess oxidative stress, anti-oxidant capacity and anti-oxidant status. The protein expression of the RISK and SAFE pathways were analysed by western blotting using both phospho-specific and total antibodies. Results and Discussion Treatment with Doxorubicin caused a time- and dose-dependent decrease in cell viability in both cell lines and this was accompanied by an increase in apoptosis. In the H9c2 cardiomyocytes, treatment with 0.2 μM Doxorubicin yielded significant levels of TNF-α after 120 hours and we can speculate that these low levels partially protected the cells from the toxic effects of Doxorubicin by activating the SAFE pathway, since both Jak2 and STAT3 were phosphorylated at this concentration. Treatment with 1 μM Doxorubicin caused a larger and biphasic pattern of TNF-α release, which may have then contributed to the decrease in cell viability, since the SAFE pathway was not activated at this concentration. Akt was phosphorylated during the first 72 hours of treatment with the low dose of Doxorubicin, but chronic treatment prevented this phosphorylation. While Erk1/2 was not phosphorylated at all at the low dose of Doxorubicin, neither Akt nor Erk1/2 was phosphorylated at the high dose and their inhibition may contribute to the cardiotoxic effects of Doxorubicin. In the C2C12 myotubes, a significant amount of TNF-α was produced after 120 hours of treatment with the low dose of Doxorubicin. Treatment with the high dose of Doxorubicin induced significant TNF-α production at every time point. While STAT3 was phosphorylated at the serine residue after treatment with the low dose of Doxorubicin, treatment with the high dose induced phosphorylation at the tyrosine residue in a time-dependent manner. p-Jak2 expression was significantly down-regulated at both concentrations of Doxorubicin, suggesting that STAT3 proteins can by-pass activation by Jak2. The Erk1/2 leg of the RISK pathway was also not activated for the majority of the treatment period, however, p-Akt expression was increased at the low concentration of Doxorubicin relative to total Akt expression. Conclusion These observations indicate that treatment with Doxorubicin causes a severe, dose-dependent loss in viability which is likely to mediated by high concentrations of TNF-α (induced by high concentrations of Doxorubicin) and down-regulation of protective signaling pathways. TNF-α may confer partial protection at low concentrations by activating the SAFE pathway. However, activation of the SAFE pathway could not provide sufficient protection from Doxorubicin, most probably because the RISK pathway was not simultaneously activated. Our results also clearly highlight the differences between acute and chronic treatment since a single high dose of Doxorubicin produced vastly different responses to cumulative treatment with a low dose. Before one can extrapolate these results into the clinical setting, further research is required to provide a better understanding of the RISK and SAFE pathways and whether stimulation thereof will provide a protective effect. In addition, although our study has shown that TNF-α is produced in response to Doxorubicin treatment, its true role, whether beneficial or detrimental, remains to be determined.

AFRIKAANSE OPSOMMING: Inleiding Die ontdekking van Doksorubisien (DOKS) in die 1960’s het die oorlewingsyfer van kankerpasiënte drasties verhoog, maar DOKS-gebruik gaan egter ook gepaard met dosis-afhanklike kardiotoksisiteit. Terwyl die literatuur grootliks fokus op akute kardiotoksisiteit, wat minimaal en algemeen omkeerbaar is, hou kroniese kardiotoksisiteit ‘n ernistige bedreiging vir kankeroorlewendes in, aangesien dit kan lei tot dilatiewe kardiomiopatie, kongestiewe hartversaking, en selfs dood. Die spesfikieke meganismes wat bydrae tot kardiotoksisiteit is tans steeds onbekend, maar oksidatiewe stres-geinduseerde miokardiale skade en apoptose word beskou as hoof bydraende faktore. Reperfussie skade, ook gekaraktiseer deur die teenwoordigheid van oksidatiewe stres en apoptose, kom voor as gevolg van die herstel van bloedtoevoer na ‘n isgemiese hart. Om die skade te minimaliseer word voor- en nakondisionerings tegnieke geïmplimenteer wat die RSHK (Reperfussie Skade Herwinnings Kinase) en OAFV (Oorlewerings Aktiverings Faktor Versterkings)-weë aktiveer. Die RSHK weg maak gebruik van pro-oorlewings kinases Erk1/2 en Akt, terwyl die TNF-α geaktiveerde OAFV weg Jak2 en STAT3 betrek. Aangesien beide reperfussie skade en DOKS-geinduseerde kardiotoksisiteit soortgelyke eienskappe deel, is die doel van hierdie studie om vas te stel of die RSHK en OAFV-weë geaktiveer word in langtermyn DOKS behandeling. Boonop is nog ‘n doel van hierdie studie om vas te stel of TNF-α geproduseer word tydens behandeling, aangesien die rol daarvan in DOKS-geinduseerde kardiotoksisiteit steeds onbekend is. Metodes H9c2 kardiomiosiet en gedifferensieerde C2C12 miobuise was daagliks behandel met toenemende konsentrasies van Dox vir 120 ure. Die effekte van DOKS op sel lewensvatbaarheid, apoptose en nekrose is onderskeidelik ondersoek deur middel van die MTT, Caspase-Glo® 3/7 en LDH toetse. TNF-α produksie is bepaal deur van die Quantikine® toets gebruik te maak, en verskeie metodes is gebuik om die oksidatiewe stres, anti-oksidantkapasiteit en anti-oksidantstatus te bepaal. Die proteïenuitdrukking van die RSHK (Erk1/2 en Akt) en OAFV (Jak2 en STAT3) weë was ontleed deur middel van westerse afklattingstegniek deur van beide fosfospesifieke en totale teenliggaampies gebruik te maak. Resultate en Bespreking Behandeling met DOKS het ‘n tyd en dosis-afhanklike afname in sel lewensvatbaarheid in beide sellyne veroorsaak, wat gepaard gegaan het met ‘n toename in apoptose. In die H9c2 kardiomiosiete, het ‘n lae DOKS dosisbehandeling (0.2 μM) betekenisvolle vlakke van TNF-α na 120 uur opgelewer en ons kan spekuleer dat hierdie lae vlakke gedeeltelik die selle van die toksiese effekte van DOKS deur die aktivering van die OAFV weg beskerm het omrede beide Jak2 en STAT3 by hierdie konsentrasie gefosforileer is. Die hoë DOKS dosis (1 μM) het ‘n groter en bifasiese patroon van TNF-α vrystelling vertoon, wat kon bydra tot die DOKS-geinduseerde afname in sel lewensvatbaarheid. Akt is gedurende die eerste 72 uur van behandeling gefosforileer met die lae DOKS dosis, maar kroniese behandeling het hierdie fosforilering verhoed. Terwyl Erk1/2 glad nie gefosforileer is by die lae DOKS dosis nie, is nie Akt of Erk1/2 by die hoë dosis gefosforileer nie, en kan hierdie inhibering bydrae tot die kardiotoksiese effekte van DOKS. In die C2C12 miobuise, is ‘n betekenisvolle hoeveelheid TNF-α na 120 uur van behandeling geproduseer by die lae DOKS dosis. Behandeling met die hoë DOKS dosis het betekenisvolle TNF-α produksie geinduseer by elke tydspunt. Terwyl STAT3 gefosforileer is by die serienresidu na behandeling met die lae DOKS dosis, het behandeling met die hoë dosis fosforilering by die tirosienresidu op ’n tydsafhanklike wyse plaasgevind. p-Jak2 uitdrukking was betekenisvol verminder by beide DOKS konsentrasies, wat aanduidend is dat die STAT3 proteïene nie geaktiveer hoef te word deur Jak2 nie. Die Erk1/2 been van die RSHK weg is ook nie geaktiveer gedurende die oorhoofse behandelingstydperk nie, alhoewel, p-Akt wel uitgedruk is by die lae konsentrasie van DOKS relatief tot die totale Akt uitdrukking. Gevolgtrekkings Die resultate van hierdie studie toon dat DOKS-behandeling tot ‘n dosis-afhanklike verlies in sel lewensvatbaarheid lei. Hierdie effek word waarskynlik bemiddel deur die teenwoordigheid van hoë konsentrasies TNF-α, en ook die afregulering van die beskermende seinweë. TNF-α kan moontlik gedeeltelike beskerming bied by lae konsentrasies deur aktivering van die OAFV weg. Die aktivering van die OAFV weg kon egter nie voldoende beskerming teen DOKS bied nie; moontlik as gevolg van die afwesigheid van die gelyktydige RSHK weg aktivering. Ons resultate vertoon die verskille tussen die akute en kronies behandeling aangesien ‘n enkele hoë-dosis van DOKS, in vergelyking met ‘n kumulatiewe lae-dosis, grootliks verskillende resultate opgelewer het. Voordat hierdie resultate klinies verder ondersoek kan word is verdere navorsing nodig om TNF-α en die RSHK en OAFV-weë beter te verstaan, en om vas te stel of stimulering van hierdie seinoordragpaaie ‘n beskermende effek teweeg sal bring.

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