Investigating the effects of diet-induced obesity on acute Doxorubicin-induced cardiotoxicity in a tumour-bearing model

Bock, Bianca Annina (2019-04)

Thesis (MSc)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT: Introduction: Obesity is a well-known global pandemic that is fundamentally caused by an energy imbalance between calories consumed and calories expended. A major consequence to obesity are metabolic perturbations that are accompanied by insulin insensitivity and central obesity. Not only, but also are these factors associated with metabolic syndrome, they increase the risk of developing cardiovascular disease. Recently, obesity has been linked to the development of cancer, particularly breast and prostate cancer. While numerous mechanisms have been proposed, by far the biological mechanisms that has received much attention include insulin, insulin-like growth factor-1 (IGF-1), sex hormones and adipokines. While research on the mechanisms underlying obesity and its relationship with cancer are still in the early stages, understanding the processes of carcinogenesis in obesity-related cancers may lead to the development of treatments and better preventative strategies. Interestingly, the use of doxorubicin (DOX), a potent chemotherapeutic agent, is limited by its cumulative, dose- dependent toxicity that can lead to the development and progression of congestive heart failure. Considering that these inflammatory diseases have mainly been studied in isolation, there are very few, if any studies that have investigated cancer and cardiotoxicity in the context of obesity, to better understand the complex relationship that these diseases have with one another and how cardiovascular function is adversely affected in this scenario. Therefore, this study hypothesized that cancer in an obese environment increases the risk of developing early-stage DOX-induced cardiotoxicity. Methods: Three-week-old female C57BL6 mice were acclimatized and randomly placed on either a high (H) fat diet or standard (S) diet for eight weeks to induce obesity. After eight weeks on the diet, these groups were further divided into two more groups were half of the mice in S and in H groups were inoculated with EO771 breast cancer cells and the others were not (non-tumour control group). Once tumours were palpable, animals were divided into more groups were some received DOX treatment. Each animal on treatment received three intraperitoneal injections of 4 mg/kg DOX resulting in a cumulative dose of 12 mg/kg. Animals remained on their respective diets for the duration of the study and were monitored daily. Four days after the last injection, animals were weighted, euthanized and their hearts excised. The blood collected was used to analyse circulating cytokines, adipokines and other markers of cardiovascular damage, while heart tissue was preserved in formalin for histological staining, or snap frozen for biochemical analyses including oxidative stress and apoptosis. Results: Animals exposed to the high fat diet gained significantly more weight by the end of the study (29.24 ± 1.99 g, p<0.0001) versus those on the standard diet (19.72 ± 0.52 g). Not only did these animals display increased gonadal adiposity, circulating levels of glucose and leptin were also elevated. Furthermore, this group of animals showed a decreased heart-to-body weight ratio and fibrosis when compared to their control counter parts. In the presence of a tumour (T), there was a considerable decline in body weight in both diet groups (S+T and H+T) when compared to the diet groups (S and H) alone. This study also observed a decrease in body weight when group S+T was compared to group H+T. Interleukin-6 (IL-6) levels were significantly augmented in group H+T (64.95 ± 11.62 pg/ml, p<0.01) versus group H (24.17 ± 5.59 pg/ml). While no major histological changes were observed in the high fat diet fed animals (H and H+T), interstitial congestion within the heart tissue and reduced fibrosis was evident in the standard fed group with a tumour (1.23±0.20%, p<0.001) when compared to the standard diet fed animals alone (2.40 ± 0.23%). When DOX (D) treatment was introduced to animals that received either diet, the body weight of animals fed the high fat diet (H+D) (25.02 ± 0.84 g, p<0.001) remained significantly higher than the animals fed the standard diet in the presence of DOX (S+D) (19.05 ± 0.23 g). Moreover, body weight loss was also observed when the high fat diet alone group (H) (29.24 ± 1.99 g) was compared to the high fat diet animals with tumours and treated with DOX (H+T+D) (21.30 ± 1.25 g, p<0.0001). This group of animals (H+T+D) also displayed significantly decreased adiposity and leptin levels, however the IL-6 concentration was elevated (126.90 ± 34.92 pg/ml, p<0.05) when compared to group H alone (24.17 ± 5.59 pg/ml). There was no evidence of cardiovascular damage or apoptotic cell death. Discussion and Conclusion: It is clear from the results above that a diet high in fat contributes to the development of obesity and the dysregulation of adipokines inducing an inflammatory state. However, this inflammatory response did not have a major impact on cardiovascular health. When the complication of cancer arises in the context of obesity, the inflammatory state is aggravated due the rise in the pro-inflammatory cytokine IL-6. Although cancer is known to result in significant weight loss, in this context, the loss of weight, particularly fat mass may be initially viewed as beneficial. However if this loss of weight is sustained over an extended period of time, detrimental effects become evident. When DOX therapy was administered in this co-morbid state, it too contributed to the decrease in body weight, adiposity and leptin concentrations, while IL-6 levels remained elevated. It is therefore possible that the weight loss observed in this study may be attributed to the stimulation of degradation pathways as these pathways are known to be triggered by inflammation and also DOX. While this study did not demonstrate considerable alterations in myocardial tissue, possibly due to the model not being severe enough to induce such changes in a short period oftime, this study is the first to our knowledge to develop a co-morbid model of obesity and cancer with chemotherapy.

AFRIKAANSE OPSOMMING: Inleiding: Vetsug is ‘n welbekende globale pandemie wat fundamenteel veroorsaak word deur ‘n energie wanbalans tussen kalorieë wat ingeneem en verbruik word. ‘n Hoofnagevolg van vetsug is metaboliese wysigings wat met insulienweerstandigheid en sentrale vetsugtigheid gepaard gaan. Nie alleen is hierdie faktore verwant aan die metaboliese sindroom nie maar dit verhoog ook die risiko vir die ontwikkeling van kardiovaskulêre siekte. Vetsugtigheid is onlangs verbind met die ontwikkeling van kanker, en meer spesifiek bors en prostaatkanker. Terwyl verskeie meganismes voorgestel is, is die mees bekende insulien, insulienagtige groeifaktor 1 (IGF-1), geslagshormone en adipokiene. Hoewel navorsing nog in ‘n vroeë stadium is om die meganismes wat onderliggend vetsug met kanker te verbind, is dit noodsaaklik om die prosesse van karsinogenese in vetsug verwante kankers te verstaan om sodoende behandelingsopsies asook voorkomende strategieë te ontwikkel. Die gebruik van doksorubisien (DOX), ‘n kragtige chemoterapeutiese middel, word beperk deur die kumulatiewe dosis-afhanglike toksisiteit wat aanleiding kan gee tot kongestiewe hartversaking. Meeste van hierdie inflammatoriese siektes is in isolasie bestudeer en daar is weinig, indien enige studies wat kanker en kardiotoksisiteit in die konteks van vetsugtigheid bestudeer het, om sodoende die komplekse verband wat hierdie siektes met mekaar deel, en hoe kardiovaskulêre funksie negatief hierdeur geaffekteer word te verstaan. Hierdie studie hipotetiseer dat kanker in ‘n vetsugtige omgewing die risiko vir die ontwikkeling van vroeë stadium DOX-geinduseerde kardiotoksisiteit verhoog. Metodes: Drie-week oue vroulike C57BL6 muise is geklimatiseer en ewekansig verdeel in hoë vetdieet (H) of standaarddieet (S) groepe vir agtweke om sodoende vetsugtigheid te induseer. Hierdie groepe is verder in twee verdeel waar een groep met E0771 borskankerselle geinokuleer is en die ander groep as kontrole gedien het. Die diere was verder verdeel in twee groepe die oomblik toe die gewasse voelbaar was met een groep wat daarna DOX ontvang het. Elke dier op behandeling het drie intraperitoneale inspuitings van 4 mg/kg DOX ontvang in ‘n kumulatiewe dosis van 12 mg/kg. Die diere is op die groepdieete gehou en daagliks gemonitor oor die verloop van die studie. Vier dae na die finale inspuiting is die diere geweeg gevolg deur genade dood waarna die harte gedisekteer is. Die bloed is versamel om sirkulerende sitokiene, adipokiene en merkers van kardiovaskulêre skade te ondersoek, terwyl die hart in formalien gepreserveer is vir histologiese kleuring of geklampvries vir biochemiese analise insluitend oksidatiewe stres en apoptose. Resulte: Die diere wat blootgestel is aan die hoë vetdieet het betekenisvolle gewigstoename teen die einde van die studie (29.24 ± 1.99 g, p<0.0001) vergeleke met die standaarddieet (19.72 ± 0.52 g) getoon. Nie net het die diere ook verhoogde gonodale adipositeit gelewer nie, maar sirkulerende glukose en leptien is ook verhoog gewees. Verder het hierdie groep diere verlaagde hart-tot-liggaamsgewigverhouding en fibrose vertoon teenoor die kontrole groep. In die teenwoordigheid van die tumor (T) was daar ‘n besondere afname in liggaamsgewig in beide die dieetgroepe (S+T and H+T) teenoor die standaardgroepe alleen (S and H). Hierdie studie het ‘n afname in liggaamsgewig tussen groep S+T teenoor groep H+T waargeneem. Interleukien-6 (IL-6) vlakke het betekensivol verskil in groep H+T (64.95±11.62 pg/ml, p<0.01) teenoor groep H (24.17±5.59 pg/ml). Terwyl geen histologiese veranderinge waargeneem is in die hoë vetdieet diere nie (H en H+T), is interstisiëele kongestie en verlaagde fibrose in die standaarddieet groep met die tumor waargeneem (1.23±0.20%, p<0.001), vergeleke met die standaarddieet groep diere (2.40±0.23%). Tydens die aanvang van DOX (D) behandeling is daar in beide dieetgroepe op die hoë vetdieet (H+D) (25.02±0.84 g, p<0.001) ‘n betekenisvolle verhoging waargeneem vergeleke met die standaarddieet groep in die teenwoordigheid van DOX (S+D) (19.05±0.23 g). Liggaamsgewig verlies is waargeneem in die hoë vetdieet alleen groep (H) (29.24±1.99 g) teenoor die hoë vetdieet groep diere met die tumor en wat met DOX behandel is (H+T+D) (21.30±1.25 g, p<0.0001). Hierdie groep diere (H+T+D) het ook betekensivolle verlaagde adipositeit en leptienvlakke vertoon hoewel die IL-6 konsentrasie verhoog was (126.90 ± 34.92 pg/ml, p < 0.05) vergeleke met die H groep alleenlik (24.17 ± 5.59 pg/ml). Daar is geen bewyse vir kardiovaskulêre skade of apoptiese seldood nie. Bespreking en gevolgtrekking: Dit is duidelik uit hierdie resultate dat ‘n hoë vetdieet bydra tot the ontwikkeling van vetsug en die disregulering van adipokiene en die inflammatoriese toestand. Hierdie inflammatoriese respons het nie ‘n groot impak op kardiovaskulêre gesondheid gehad nie. Wanneer kanker in ‘n vetsugtige konteks ondersoek word, het die inflammatoriese toestand vererger as gevolg van die toename in die pro-inflammatoriese sitokien IL-6. Hoevewel kanker gewoontlik met gewigsverlies geassosieer word, kan die gewigsverlies wat in hierdie konteks waargeneem is, veral vetmassa, aanvanglik voordelig wees. Indien hierdie gewigsverlies oor ‘n langer tydperk gehandhaaf word kan dit as nadelig en gevaarlik beskou word. Wanneer DOX terapie toegedien word in die ko-morbiede stadium het dit self bygedra tot die verlies aan liggaamsgewig, adipositeit en leptienkonsentrasies, terwyl IL-6 vlakke verhoog was. Dit is dus moontlik dat die gewigsverlies wat in hierdie studie waargeneem is bydraend tot die stimulering van degraderingspadweë, en dat hierdie wëe ook deur inflammasie en DOX gestimuleer is. Alhoewel hierdie studie nie merkbare veranderinge in die miokardiale weefsel kon demonstreer nie, omrede die model in ‘n relatief kort periode nie die skade kan veroorsaak nie, het die studie wel daarin geslaag om ‘n nuwe ko-morbiede model van vetsug, kanker en chemoterapie te beskryf.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/106156
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