A matter of life or death : autophagy in the context of prolonged doxorubicin therapy

Adonis, Muneeb (2019-04)

Thesis (MSc)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT: Introduction Doxorubicin (DOX) is an effective treatment against a variety of cancers, and thus remains a commonly used chemotherapeutic drug. The chief side effect of DOX treatment is cardiotoxicity. The precise mechanisms by which DOX induces cardiotoxicity are unknown. One of the most accepted mechanisms is the excess production of ROS. The structure of DOX allows it to undergo redox cycling and form DOX-iron complexes, both of which generate free radicals. DOX-induced oxidative damage is more prevalent in the heart than other organs. As the pathogenesis of cardiotoxicity appears to be mediated by oxidative stress, it seems as if the most effective treatment would be antioxidant therapy. Antioxidant therapy has proved unsuccessful. Autophagy is a catabolic process which allows a cell to remove cytoplasmic components. Components regarded as surplus or defective may be removed to ensure the survival of the cell. There have been numerous studies conducted to evaluate the relationship between DOX and autophagy. Many of these studies have concluded that DOX treatment does affect autophagy. Although there is literature to support both the up and downregulation of autophagy. We hypothesis that during the development of DOX-induced cardiotoxicity autophagy is downregulated. Attenuation of this downregulation during DOX therapy will attenuate the effects of cardiotoxicity. Methodology Three-week-old male black 6 (C57BL/6) mice were split into six groups; vehicle control, DOX, rapamycin, a starvation, DOX and rapamycin combination and DOX and starvation group. The DOX group received 2mg/kg DOX weekly for a total of eight weeks, resulting in a cumulative dose of 16mg/kg. The rapamycin group received 2mg/kg rapamycin weekly. The DOX and rapamycin group received rapamycin 30 minutes prior to receiving DOX. The starvation group was starved from food for 24 hours each week but were allowed free access to drinking water. The DOX and starvation group were starved for 24 hours prior to receiving DOX. A week after the final treatment, mice were euthanised. The whole hearts were harvested and sectioned, roughly into halves. One half of each was stored in formaldehyde solution for histological analysis, the other was frozen in liquid nitrogen for biochemical analysis. H&E staining was performed to assess tissue morphology and cardiomyocytes area. Picrosirius red staining was performed to assess fibrosis. Fluorometry was done to assess oxidative stress via GSH, GSSG and CDs levels. Western blots were performed to assess expression of caspase 3 and LC3. A Kolmogorov- Smirnov normality test was done to test for a normal distribution. Appropriate statistical tests were applied. P-values were considered significant when P < 0.05. Results Differences in body mass first appeared at week 5, with both DOX+ Rapa and DOX+ starve weighing less than control. Differences then appeared (at week 6) between DOX and control, with DOX weighing less than control. These differences continued until the end of the study. Histology showed cardiomyocyte area was decrease in the DOX group (97.4± 4.1) the control (112.6± 4.3). There was also an increase in fibrosis in the DOX group (2.07± 0.22) compared to the control (1.05± 0.18) and the DOX+ starve group (1.21± 0.18) had less fibrosis than DOX alone. Biochemical analysis showed autophagy was decreased in the DOX group (0.3733± 0.0683) compared to the control group (1.1420± 0.2262). However, the DOX+ Rapa (0.4344± 0,0543) and DOX+ starve (0.4818± 0.1240) groups also had decreased autophagy compared to the control group. Discussion The histological analysis confirms cardiotoxicity as DOX caused cardiac damage. Biochemical analysis was less straightforward. Oxidative stress markers were tested for too long after levels may have been detectable. While this provides no oxidative stress data, it suggests the cardiotoxicity was chronic rather than acute, as cardiac damage persisted after the stress had ended. Apoptotic data was inconclusive. DOX treatment caused a decrease in autophagy. Meaning, as hypothesised, this could be the target of an adjuvant therapy. The adjuvant treatment groups showed some improvement when compared to the DOX group. Both the DOX+ Rapa and DOX+ starve group showed histological improvement. The LC3 data however showed, the adjuvant therapies did not upregulate autophagy. Neither DOX+ Rapa nor DOX+ starve showed any increases in autophagy when compared to DOX. Due to the lack of attenuation of autophagic dysregulation, the reason for the protective effect of the adjuvant therapy is a mystery. Regardless, this study suggests that autophagic upregulation a potential adjuvant therapy.

AFRIKAANSE OPSOMMING: Inleiding Doksorubisien (DOX) dien as ‘n effektiewe behandelingsmiddel in onkologie en sodoende ‘n fundamentele chemoterapeutiese middel wat teen ‘n verskeidenheid kankers gebruik word. Die kliniese gebruik hiervan word beperk deur die kumulatiewe dosis afhangklike newe effekte wat maande, jare en selfs dekades na behandeling aanleiding kan gee tot die ontstaan van hartversaking. Daar word voorgestel dat kardiotoksisiteit, wat die hoof newe effek is wat met doksorubisien toediening geassosieer word, gerig is op oormatige ROS produksie wat ‘n toestand van oksidatiewe stres veroorsaak en gevolglik seldood as ‘n afstroom effek veroorsaak. Dit is verder bekend dat die chemiese struktuur van DOX betrokke is in resirkulering en die vorming van DOX-yster komplekse wat beide vryradikale kan produseer. Omrede die hart energie moet produseer om aan sy funksionering te kan voldoen in hierdie hoë oksidatiewe omgewing met lae anti-oksidant beskerming, word regenerasie kapasiteit ingeperk. Dit is dus verstaanbaar hoekom hierdie orgaan vatbaar is vir oksidatiewe stress. Hoewel kardiotoksisiteit geklassifiseer kan word as akute- en chronies stadium, is daar beperkte behandelingsopsies of ondersteuningsterapie om hierdie vertraging van newe effekte te voorkom. Outofagie, ‘n evolusionêre konserveringsproses van proteïendegradasie, het voorheen in verskeie kardiovaskulêre patologieë beskerming getoon. Daar is ook verskeie teenstrydige bewyse in die literatuur oor die rol van DOX en outofagtiese aktiwiteit; dit is nie duidelik of die onaktiewiteit daarvan bydra tot kardiotoksisiteit en/of die aktiwiteit beskerming bied nie. Hierdie studie ondersoek dus die potensiële beskermings effekte van outofagiese opregulering in ‘n rotmodel met verlengde DOX behandeling. Metodologie: Nadat etiese goedkeuring vir die studie verleen is, is drie week oue manlike swart 6 (C57BL/6) muise ontvang en ewekansig verdeel in ses eksperimentele groepe na klimatisering. Hierdie groepe sluit in: ‘n kontrole (draer), rapamisien, uithongering, DOX en kombinasies van DOX met rapamisien of uithongering. Terwyl die DOX en rapamisien groepe 2 mg/kg DOX of rapamisien weekliks ontvang het, is die uithongeringsgroep 24 uur weerhou van voedsel. In die kombineringsgroep is rapamisien 30 minute voor DOX behandeling toegedien, wat vooraf met ‘n 24 uur uithongeringsperiode vooraf gegaan is voordat chemoterapeutiese behandeling begin is. Die behandelingsprotokol is vir agtweke volhou met alle toedienings intraperitoneaal gelewer. Een week na die finale toediening is die muise met behulp van eutanasie dood gemaak waarna die harte verwyder is. Een helfte van die hart is in formaldehiedoplossing vir histologiese evaluasie geberg, en die ander helfte gevries in vloeibare stikstofvir biochemiese analises. Om die morfologiese wysigings te ondersoek asook ontwikkeling van fibrose is ‘n H&E en Picrosirius rooikleuring uitgevoer. Anti-okisdant status en oksidatiewe skade is bepaal deur veranderinge in die gereduseerde versus geokssideerde glutatioonverhouding en die teenwoordigheid van gekonjugeerde diëene onderskeidelik. Ten slotte is apoptose bepaal deur die uitdrukking van kaspase-3 vlakke. Gepaste statistiese toetse is gebruik waar p < 0.05 as betekenisvol beskou is. Resultate Verskille in die liggaamsmassa is beduidend teen die vyfde week met DOX behandeling en die outofagiese opregulering het geen bydrae gelewer tot hierdie verlies aan massa nie. Geen verandering in miokardiale massa is waargeneem by enige van die behandelingsgroepe nie. Daar is wel betekenisvolle fibrose (2.07±0.22%, p < 0.001) met ‘n afname in miobrillêre area (97.41±4.1 M2, p < 0.01) in die DOX behandelings groepe versus die kontrole (1.05 ±0.18% fibrose, 112.6 ±4.32 M2) opgemerk. Vergeleke met die DOX groep, het die kombineringsgroep verlaagde fibrose in uithongering getoon (1.21±0.18%, p < 0.01) en ‘n matige toename in miofibrillêre area is waargeneem. Verbasend is daar geen veranderinge waargeneem in die oksidatiewe stresparameters en induksie van apoptose nie. Hierdie studie het suskesvol demonstreer dat DOX ‘n kragtige inhiberings effek op outofagie het omrede beide intervensies wat gebruik is om die aktiwiteit te stimuleer onsuksesvol was om hierdie toestand te handhaaf in die teenwoordigheid van DOX. Bespreking Hierdie studie het die terapeutiese potensiaal van verhoogde outofagie deur farmakologiese (rapamisien) en ‘n fisiologiese (uithongering) benadering ondersoek in ‘n poging om die newe effekte wat met DOX terapie in die hart geassosiëer word te verlaag. Hoewel die model wat hier gebruik is uniek is en wel sekere eienskappe vertoon het, hoofsaaklik fisies (liggaamsmassa) en struktureel (miofibrillêre area en fibrose); het die biochemiese parameters (oksidatiewe stres en apoptose) onoortuigende resultate opgelewer. Hoewel die resultate van ons studie veronderstel dat strukturele modifikasies aanvang neem voordat biochemiese veranderinge onstaan, kan daar geen defnitiewe afleidings gemaak word oor die die impak van terapeutiese intervensie in hierdie scenario nie.

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