The effect of melatonin treatment on doxorubicin-induced skeletal muscle atrophy within a cancer model

Isaacs, Ashwin Wayne (2018-12)

Thesis (PhD)--Stellenbosch University, 2018.

Thesis

ENGLISH ABSTRACT: Background and Aim: Skeletal muscle atrophy is a major concern in patients suffering with malignancy. Chemotherapeutic agents, such as doxorubicin (DOX), can further exacerbate this loss of skeletal muscle. Although many cancer patients on chemotherapeutic agents suffer from this condition, there are no therapies routinely used to moderate muscle atrophy. The aim of the study was to investigate whether melatonin (MLT) can attenuate doxorubicin‐induced skeletal muscle and myotube atrophy in an in vivo rodent model of breast cancer as well as in an in vitro model of DOXinduced myotoxicity respectively. The safe and cost‐effective role of melatonin as a possible therapy to limit the burden of doxorubicin‐induced muscle toxicity in cancer patients serves as rationale for the in vivo study and the in vitro study allows for the exploration of more invasive mechanistic aspects using the cell lines, which would not be possible when viewing excised tissue. Methods: Female Sprague‐Dawley rats were inoculated with LA7 cancer cells and were randomly assigned to six groups: Control, Tumour control (TCON), Vehicle control (VEH), MLT, DOX and DOX + MLT (DM). Prophylactic treatment of MLT (6 mg/kg) was administered in drinking water daily and rats received three intraperitoneal injections of DOX (4 mg/kg, 3 times at 3‐day intervals). Following sacrifice blood samples (whole blood counts) and skeletal muscle tissue were collected for histological, immunoblot, antioxidant capacity and immunofluorescence analyses. Furthermore, C2C12 myoblasts grown to confluency and differentiated into myotubes were pretreated with MLT (50 nM) for 48h followed by DOX treatment (0.8 μM) for 24h. The effect of MLT treatment on C2C12 myotube diameter, mitochondrial reactive oxygen species (mtROS) production, sirtuin levels and autophagy activity was then assessed. Results: DOX treatment significantly reduced animal weight (279.1 ± 21.34 g vs. 222.2 ± 20.40 g, p˂0.0001) compared to DM weight (281.5 ± 7.11 g vs. 284.0 ± 6.53 g) and gastrocnemius muscle weight (1.4 ± 0.13 g vs. 0.99 ± 0.076 g, p˂0.0001) and cross sectional area (CSA), while increasing markers of muscle degradation compared to MLT treated groups. Serum myoglobin levels were significantly elevated in the DOX group compared to the DM group (572.6 ± 444.19 ng/mL vs. 218.2 ± 83.66 ng/mL, p˂0.0001); while, white & red blood cell counts (WBC & RBC) were significantly decreased in the DOX group compared to the MLT treated groups respectively (2.06 ± 1.59 x 109L‐1 vs. 4.13 ± 1.56 x 109L‐1 & 4.00 ± 1.52 x 1012L‐1 vs. 5.66 ± 1.03 x 1012L1, p˂0.0001). Furthermore, MLT treatment significantly increased intramuscular antioxidant capacity, mitochondrial biogenesis and satellite cell number. In vitro DOX treatment resulted in increased myotube atrophy, mitochondrial ROS levels and these effects were significantly reduced with MLT pre‐treatment. Discussion: The improvement in animal weight, muscle to body weight ratio, muscle CSA as well as the reduction in myoglobin levels in the treatment groups compared to the DOX group indicate that MLT protects against DOX‐induced atrophy. Moreover, MLT pre‐treatment improved circulating levels of WBC & RBC compared to the DOX only group and attenuated skeletal muscle atrophy by reducing cell apoptosis and increasing satellite cell number suggesting that MLT assists with muscle repair. The in vitro study indicated that DOX‐induced myotube atrophy was preceded by increases in mitochondrial ROS. Conclusion: Results indicate that pre‐treatment with exogenous MLT protects against skeletal muscle wasting induced by DOX in a pre‐cachectic tumour‐bearing rat model.

AFRIKAANSE OPSOMMING: Agtergrond en Doel: Skeletspier atrofie is ‘n groot bekommernis van pasiënte wat aan kanker ly. Chemoterapie soos doxorubicin (DOX) kan skeletspierverlies nog verder vererger. Alhoewel heelwat kankerpasiënte wat chemoterapie kry, hierdie toestand onder lede het, is daar nog geen terapie wat normaalweg toegedien word om dit te verlig nie. Die doel van hierdie studie was dus om te bepaal of melatonien (MLT) doxorubicin‐geïnduseerde skeletspieratrofie in ‘n in vivo rotmodel van borskanker sowel as in ‘n in vitro model van DOX‐geïnduseerde miotoksisiteit kan teenwerk. Die veilige en koste effektiewe rol van melatonien as ‘n moontlik terapeutiese middel om doxorubicin‐geïnduseerde spiertoksisiteit in kankerpasiënte te verminder, dien as rasionaal vir die in vivo studie en die in vitro studie is gedoen om meganistiese aspekte van die werking van die terapie meer in diepte te ondersoek. Metodes: Vroulike Sprague‐Dawley rotte is met LA7 selle geïnokuleer en is in ses groepe verdeel: Kontrole, Tumor Kontrole (TCON), Oplosmiddel Kontrole (VEH), MLT, DOX en DOX + MLT (DM). Die rotte is profilakties met MLT (6 mg/kg), wat daagliks in hul drinkwater toegedien is, behandel. Die rotte het ook drie intraperitoneale DOX (4 mg/kg, 3 keer met 3 dae intervalle) inspuitings ontvang. Na die eksperimentele tydperk, is bloedmonsters (heelbloedtellings) en skeletspier versamel vir histologie, immunoblot, antioksidant kapasiteit en immunofluoresensie bepalings. Verder is C2C12 mioblaste gedifferensieer in miobuise en met MLT (50 nM) behandel vir 48 uur voordat 0.8 M DOX toegedien is vir 24 uur. Die effek van MLT in C2C12 miobuise is bepaal deur die miobuis deursnit, mitokondriale reaktiewe suurstof spesie (mtROS) produksie, sirtuinvlakke en autofagie aktiwiteit te bestudeer. Resultate: DOX behandeling het die gewig van die diere insiggewend verminder (279.1 ± 21.34 g vs. 222.2 ± 20.40 g, p˂0.0001) asook die gastrocnemius spiermassa (1.4 ± 0.13 g vs. 0.99 ± 0.076 g, p˂0.0001) en deursnitarea (CSA) in vergelyking met die DM groep (281.5 ± 7.11 g vs. 284.0 ± 6.53 g), terwyl merkers van atrofie verhoog is in vergelyking met die MLT groepe. Serum myoglobien vlakke is insiggewend in die DOX groep verhoog in vergelyking met die DM groep (572.6 ± 444.19 ng/mL vs. 218.2 ± 83.66 ng/mL, p˂0.0001); terwyl die wit‐ en rooibloedseltellings (WBS & RBS) weer insiggewend verlaag is wanneer dit met die MLT groepe vergelyk is (2.06 ± 1.59 x 109L‐1 vs. 4.13 ± 1.56 x 109L‐1 & 4.00 ± 1.52 x 1012L‐1 vs. 5.66 ± 1.03 x 1012L‐1, p˂0.0001). Verder het MLT behandeling die intramuskulêre antioksidant kapasiteit, mitokondriale biogenese en aantal satellietselle insiggewend verhoog. In vitro DOX behandeling het miobuis atrofie asook mitokondriale ROS vlakke verhoog, terwyl hierdie effekte insiggewend verlaag is in die groepe wat vooraf met melatonien behandel is. Bespreking: Die toename in die diere se gewig, spier tot liggaamsgewig ratio en spierdeursnitarea asook die verlaging in mioglobienvlakke in die MLT behandelde groepe in vergelyking met die DOX groepe, bewys dat MLT die liggaam teen DOX‐geïnduseerde atrofie beskerm. Verder het MLT behandeling ook die sirkulerende vlakke van WBS & RBS verhoog in vergelyking met die DOX behandeling, terwyl apoptose verminder het en satellietselle toegeneem het, wat aanduidend is van MLT se rol in spierherstel. Die in vitro studie dui aan dat DOX‐geïnduseerde miobuis atrofie voorafgegaan is deur ‘n toename in mitokondriale ROS. Gevolgtrekking: Resultate dui aan dat vooraf behandeling met eksogene MLT, DOX‐geïnduseerde skeletspieratrofie teenwerk in ‘n tumor‐draende rot model.

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