Mitochondrial catastrophe during doxorubicin-induced cardiotoxicity : An evaluation of the protective role of melatonin.

Govender, Yogeshni (Jenelle) (2017-03)

Thesis (PhD)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: Introduction: Anthracyclines, such as doxorubicin (DXR), are among the most valuable treatments for various cancers, but their clinical use is limited due to detrimental side-effects such as cardiotoxicity. The abundance of mitochondria in cardiomyocytes closely links mitochondrial function with myocardial function. Mitochondrial dysfunction has emerged as a critical element in the development of DXR-induced cardiotoxicity. In light of this scenario, melatonin (MLT) is a potent anti-oxidant, is non-toxic, is dually oncostatic and cardio-protective, and has been shown to influence mitochondrial homeostasis and function. Both endogenously produced and exogenously administered MLT during or prior chemotherapy shows great promise in this therapeutic avenue as demonstrated in several studies. Although research support the mitochondrial protective role of MLT, the exact mechanisms by which MLT confers mitochondrial protection in the context of DXR-induced cardiotoxicity remains to be elucidated. Aims: The aim of this study was to investigate the effect of MLT on the following mitochondrial and cellular parameters: mitochondrial reactive oxygen species (ROS) production, mitochondrial membrane potential, mitochondrial fission and fusion, mitochondrial bioenergetics and biogenesis, sirtuin activity, autophagy and cell death in an in vitro model of DXR-induced cardiotoxicity. Furthermore, the effect of MLT on cardiac function and tumor growth was assessed in a tumor-bearing rat model of acute DXR-induced cardiotoxicity. Materials and Methods: H9c2 rat cardiomyoblasts were pre-treated with MLT (10 μM) for 24 hours followed by DXR treatment (3 μM) for 24 hours. Following treatment, the above mentioned mitochondrial and cellular parameters were assessed using immunoblot analysis, mitochondrial respiration analysis, flow cytometry, fluorescence microscopy and luciferase-based assays. Sprague Dawley female rats (16-18 weeks old), were inoculated with LA7 rat tumor cells. Animals received DXR (3 intraperitoneal injections of 4 mg/kg at 3-day intervals, 12 mg/kg cumulative dose) and/or received MLT (6 mg/kg) daily in their drinking water. Tumors were measured daily using digital calipers and tumor volumes calculated. Animal weights were recorded daily. Rat hearts were used to conduct isolated heart perfusions to assess cardiac function and thereafter, heart tissue was used for immunoblot analysis. Results: DXR treatment significantly increased cell death, mitochondrial ROS levels and mitochondrial fission and these effects were significantly reduced with MLT pre-treatment. Furthermore, MLT pre-treatment significantly increased mitochondrial membrane potential, mitochondrial biogenesis and cellular ATP levels reduced by DXR treatment. Cardiac output and total work performance of the heart was significantly increased in rats treated with DXR+MLT in comparison to rats treated with DXR alone. In addition, body and heart weights were significantly reduced in DXR-treated rats in comparison to DXR+MLT treated rats. Tumor volumes were significantly reduced in DXR+MLT-treated rats on Day 8 in comparison to DXR-treated rats. Discussion and Conclusion: The results obtained from the current study indicates that MLT treatment confers a cardio-protective effect by maintaining mitochondrial function, increasing cardiomyocyte survival and improving cardiac function during DXR-induced cardiotoxicity. Furthermore, MLT treatment alone suppresses the growth of tumors. The combination of DXR+MLT treatment rapidly reduced tumor growth, suggesting that MLT enhances the oncostatic activity of DXR. The unique ability of MLT to be both cardio-protective and oncostatic during DXR-induced cardiotoxicity is promising for the field of cardio-oncolocgy.

AFRIKAANSE OPSOMMING: Inleiding: Antrasikliene, soos doxorubicin (DXR), is van die mees waardevolle behandelings-opsies vir verskeie tipes kankers, maar die kliniese gebruik daarvan word deur die nadelige newe-effekte beperk, wat kardiotoksisiteit insluit. Die groot hoeveelheid mitochondria in kardiomiosiete verbind mitochondriale funksie aan kardiale funksie. Na aanleiding hiervan, is daar al bewys dat melatonien (MLT), ‘n kragtige antioksidant, nie-toksies, gelyktydig onkostaties en kardio-beskermend, mitochondriale homeostase en funksie kan beïnvloed. Dit is al in verskeie navorsing bewys dat beide endogeen geproduseerde en eksogeen toegediende MLT tydens en voor chemoterapie groot belofte as behandelings-opsie inhou. Alhoewel navorsing al die beskermende rol van MLT bewys het, moet die presiese meganisme waardeur MLT mitochondria beskerm in die konteks van DXR-geïnduseerde kardiotoksisiteit, nog bepaal word. Doelwitte: Die doel van hierdie studie was om die effek van MLT op die volgende mitochondriale en sellulêre parameters in ‘n in vitro model van DXR-geïnduseerde kardiotoksisiteit te bepaal: Mitochondriale reaktiewe suurstofspesie produksie, mitochondriale membraanpotensiaal, mitochondriale splyting en fusie, mitochondriale bio-energie produksie en biogenese, sirtuin aktiwiteit, autofagie asook seldood. Die effek van MLT op kardiale funksie en tumorgroei is ook in ‘n tumor-draende rot model van akute DXR-geïnduseerde kardiotoksisiteit geondersoek. Materiaal en Metodes: H9c2 rot kardiomioblaste is met MLT (10 M) vir 24 uur behandel voordat die met DXR (3 M) ook vir 24 uur behandel is. Na behandeling is die bo-vermelde mitochondriale en sellulêre parameters bepaal deur middel van immunoblot bepalings, mitochondriale analises, vloeisitometrie, fluoressensie mikroskopie en lusiferase-gebaseerde bepalings. Sprague Dawley vroulike rotte (16-18 weke oud) is met LA7 rot tumorselle geïnokuleer. Die rotte het DXR (3 intraperitoneale inspuitings van 4 mg/kg met 3-dag intervalle en ‘n 12 mg/kg kumulatiewe dosis) en/of MLT (6 mg/kg) daagliks in hul drinkwater ontvang. Tumore is daagliks met ‘n digitale meetpasser gemeet en tumor volumes is bereken. Die rotte is ook daagliks geweeg. Rotharte is geïsoleer en geperfuseer om kardiale funksie te bepaal en daarna is dit vir immunoblot analises gebruik. Resultate: DXR behandeling het seldood geïnduseer, mitochondriale reaktiewe suurstofspesie vlakke verhoog en het ook mitochondriale splyting veroorsaak. Hierdie negatiewe effekte van DXR is beduidend verminder wanneer die selle vooraf met MLT behandel is. MLT behandeling het ook mitochondriale membraanpotensiaal, mitochondriale biogenese en sellulêre ATP vlakke verhoog wat deur DXR behandeling verlaag was. Kardiale omset en kardiale werkverrigting van die harte was beduidend verhoog in rotte wat met beide DXR en MLT behandel is in vergelyking met harte van die DXR behandelde rotte. Verder is die liggaamsgewig en die massas van die harte ook beduidend verlaag in vergelyking met die DXR en MLT behandelde rotte. Tumor volumes is ook alreeds beduidend verlaag op dag 8 in die DXR en MLT behandelde rotte in vergelyking met die DXR behandelde rotte. Bespreking en Gevolgtrekking: Die resultate van hierdie studie bewys onteenseglik dat MLT behandeling sy kardio-beskermende effekte tydens DXR-geïnduseerde kardiotoksisiteit teweegbring deur mitochondriale funksie te handhaaf, asook om kardiomiosiet oorlewing en kardiale funksie te verbeter. Verder kan MLT behandeling op sy eie ook tumor-groei onderdruk. Die kombinasie van DXR en MLT behandeling het tumor-groei vinniger geonderdruk wat moontlik ‘n bewys is dat MLT die onkostatiese aktiwiteit van DXR kan verhoog. Hierdie unieke vermoë van MLT om beide kardio-beskermend en onkostaties tydens DXR- geïnduseerde kardiotoksisiteit te wees, maak van MLT ‘n belowende terapeutiese opsie in die veld van kardio-onkologie.

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