The potential toxic effects of chronic doxorubicin treatment on the rat pancreas and the role of ghrelin in this context

Scott, Kirsten Lee (2018-03)

Thesis (MSc)--Stellenbosch University, 2018.

Thesis

ENGLISH ABSTRACT: Introduction: Doxorubicin (DOX), is a chemotherapeutic drug that has potent anti-neoplastic actions. It is for this reason that it remains one of the most widely used chemotherapeutic agents that has led to an increase in the survival rates of cancer patients. However, DOX’s efficacy in treating a variety of cancers is a double-edged sword due to its cumulative, dose-dependent toxicity, particularly in cardiomyocytes. Since DOX’s anti-neoplastic activities are separate to the mechanisms underlying its toxicity, there is a need to investigate adjuvant therapies that do not interfere with DOX’s ability to kill cancerous cells, but have the potential to protect against its toxicity. Ghrelin, a brain-peptide commonly known for its appetite inducing and growth hormone (GH) releasing actions, has previously been shown to reduce oxidative stress, apoptosis, inflammation and fibrosis, all of which contribute to DOX-induced toxicity, in different contexts. However, over the years, literature has predominantly focused on DOX’s effects on the heart, while very few studies are available regarding DOX’s effects on the pancreas. Therefore, this study investigated the effects of DOX on the pancreas and whether ghrelin can provide protection against these effects in a model of chronic DOX-induced cardiotoxicity. Materials and Methods: Male Sprague-Dawley were randomly divided into four treatment groups. The vehicle group received 200 μl of physiological saline, the ghrelin group received 100 μg/kg three times a week, the DOX group received 2.5 mg/kg once a week, and the combination group (DOX+ghrelin) received both treatment regimens. All treatments were conducted via intraperitoneal injection over a period of eight weeks. A week after the last injection, animals were euthanised, blood was collected, and organs were harvested. After the pancreata were weighed, they were non-specifically divided into two sections, where one half was presevered in 4% formaldehyde solution for histological analysis, and the other half was snap frozen in liquid nitrogen for biochemical analysis. Serum inflammatory markers as well as pancreatic hormones insulin and glucagon were measured using a multiplex assay. General morphological changes, collagen deposition, and the number of α- and β-cells were assessed by employing H&E, Masson’s Trichrome, and immunohistochemical stains, respectively. In addition to lipid peroxidation, oxidative stress was assessed by the ORAC, SOD and glutathione assays. Finally, Western blotting was utilised to determine the expression of the apoptotic marker, cleaved caspase-3. Results: Following eight weeks of treatment, DOX significantly reduced appetite (152.95 ± 10.23 g, p<0.05) and weight gain (186.88 ± 10.35 g, p<0.0001) when compared to the saline treated animals. Ghrelin, in the presence of DOX did not significantly differ when compared to the DOX treated group. DOX caused significant collagen deposition which is an indication of fibrosis in the pancreas of these animals (4.80 ± 0.78%, p<0.0001), whereas DOX+ghrelin significantly reduced collagenous areas (2.22 ± 0.39%, p<0.001) when compared to the DOX group. Our oxidative stress analyses revealed that both the DOX (0.51 ± 0.028 μmol TE/g, p<0.01) and DOX+ghrelin (0.47 ± 0.01 μmol TE/g, p<0.05) groups considerably increased their anti-oxidant capacity when compared to the vehicle (0.37 ± 0.05 μmol TE/g). Moreover, SOD activity was significantly downregulated in both the DOX (1.49 ± 0.18 U/mg, p<0.01) and DOX+ghrelin (1.54 ± 0.12 U/mg) groups when compared to the vehicle. Cleaved caspase 3 was also elevated during DOX treatment but reduced in the combination group. No other noteworthy changes were observed in any of the other parameters measured. Discussion and Conclusion: The results of this study indicate that DOX is a cytotoxic agent that induces a loss of appetite and detrimental effects such as oxidative stress, fibrosis and cell death in pancreatic tissue. The use of ghrelin as an adjuvant treatment in this context was beneficial as weight gain was promoted, while fibrosis and cell death were reduced. Although the exact mechanisms underlying ghrelin’s orexigenic effects are still unknown, it is believed that ghrelin acts through the hypothalamic-pituitary axis to promote appetite. Ghrelin’s anti-fibrotic effects are induced through the downregulation of pro-inflammatory and pro-fibrotic cytokines, while apoptosis is prevented via the inhibition of cytochrome c leakage from the mitochondria. Since ghrelin had no effect in improving the activity of SOD, its anti-oxidant effects could not be proved in this tissue. In conclusion, while these results shed some light into understanding the mechanisms by which ghrelin counteracts DOX’s effects, further research is necessary to assess ghrelin’s potential as an adjuvant treatment regimen for DOX-induced pancreatic injury.

AFRIKAANSE OPSOMMING: Inleiding: Doksorubisien (DOX), is ‘n chemoterapeutiese middel wat sterk anti-neoplastiese werking toon. Weens hierdie rede is dit steeds een van die mees algemeen chemoterapeutiese middels wat oorlewing in kanker pasiënte verhoog. Daarbenewens, is die gebruik van DOX weens die effektiwiteit teen verskeie kankers paradoksaal weens die kumulatiewe, dosis-afhanglike toksisiteit veral in kardiomiosiete. Omrede DOX se anti-neoplastiese aktiwiteite apart staan van die onderliggende toksisiteitsmeganismes, is daar ‘n behoefte om adjuvante terapie te ondersoek wat nie met DOX se vermoë inwerk om kankerselle te vernietig nie, maar om teen die toksisiteit te beskerm. Grelien, ‘n breinpeptied wat algemeen bekend is vir sy aptytinduserende, en groeihormoon (GH) vrystellingseienskappe, is voorheen bewys om oksidatiewe stres, apoptose, inflammasie en fibrose, wat bydraes lewer tot DOX geïnduseerde tokisisteit, in verskeie kontekse, te verlaag. Geskiedkundig, fokus die literatuur meestal op DOX se effek op die hart, terwyl daar min studies die effek op die pankreas rapporteer. As gevolg hiervan, het hierdie studie die effekte van DOX op die pankreas, en of grelien beskerming kan bied teen hierdie newe-effekte in ‘n chroniese DOX geïnduseerde kardiotoksisiteitmodel. Materiale en Metodes: Manlike Sprague-Dawley rotte is ewekansig verdeel in vier behandelingsgroepe. Die draergroep het ‘n fisiologiese soutoplossing ontvang, die grelien groep, 100 μg/kg drie keer per week, die DOX groep, 2.5 mg/kg een keer per week, en die kombinasie groep (DOX+grelien) het beide behandelings ontvang. Alle behandelings is via intraperitoneale toediening oor ‘n agt-weke periode gedoen. Een week na die laaste toediening, is die diere deur middel van eutanasie doodgemaak, waarna bloedmonsters versamel, en die organe verwyder is. Nadat die pankrease geweeg is, is hulle in twee dele gedissekteer, waar die een helfte met ‘n 4% formaldehiedoplossing vir histologiese analiese voorbereid is, en die ander helfte met vloeibare stikstof bevries is vir biochemiese analieses. Serum inflammatoriese merkers, sowel as pankreatiese hormone en glukagon, is deur middel van ‘n multiplekstoetsing gedoen. Algemene morfologiese veranderinge, kollageenneerlegging, en aantal α- en β-selle is deur middel van H&E, Masson’s Trichrome, en immunohistochemiese kleuring onderskeidelik ondersoek. Addisioneel tot lipiedperoksidasie, is oksidatiewe stres deur die ORAC, SOD en glutatioontoetse ondersoek. Laastens, is Westerse kladtegniek gebruik om die uitdrukking van die apoptotiese merker, gesplyte kaspase-3 te ondersoek. Resultate: Na afloop van die agt-weke behandeling, het DOX betekenisvol aptyt verlaag (152.95 ± 10.23 g, p<0.05) asook gewigstoename (186.88 ± 10.35 g, p<0.0001) vergeleke met die soutoplossing groep. Grelien, in die teenwoordigheid van DOX, het nie aptyt en gewigstoename betekenisvol gestimuleer teenoor die DOX groep nie. DOX het ‘n beteknisvolle neerlegging van kollageen in die pankreasse van hierdie diere veroorsaak wat aantoon dat fibrose teenwoordig is (4.80 ± 0.78%, p<0.0001), terwyl DOX+grelien hierdie kollageenareas verlaag het (2.22 ± 0.39%, p<0.001) vergeleke met die DOX groep. Die oksidatiewe stresanalises toon aan dat beide DOX (0.51 ± 0.028 μmol TE/g, p<0.01) en DOX+grelien (0.47 ± 0.01 μmol TE/g, p<0.05) groepe hulle anti-oksidantkapasiteit verhoog vergeleke met die draer groep (0.37 ± 0.05 μmol TE/g). Die SOD aktiwiteit is betekenisvol afgereguleer in beide die DOX (1.49 ± 0.18 U/mg, p<0.01) en DOX+grelien (1.54 ± 0.12 U/mg) groepe vergeleke met die draer groepe. Gesplyte kaspase-3 is ook verhoog tydens DOX behandeling, maar verlaag in die kombinasie groep. Geen ander merkbare veranderinge is aangetoon in enige van die ander parameters nie. Bespreking en Gevolgtrekking: Die resultate van hierdie studie toon dat DOX ‘n sitotoksiese middel is en verlies aan aptyt met skadelike effekte soos oksidatiewe stres, fibrose en seldood in pankreatiese weefsel, induseer. Die gebruik van grelien as ‘n adjuvante behandelingsmiddel in hierdie konteks was voordelig omrede gewigstoename bevorder was, terwyl fibrose en seldood verlaag is. Hoewel die presiese meganismes van grelien se oreksigeniese effekte onbekend is, is dit moontlik dat grelien deur die hipotalamiese-pituitêre aksis werk en sodoende aptyt bevorder. Grelien se anti-fibrotiese effekte word deur die afregulering van pro-inflammatoriese en pro-fibrotiese sitokiene bewerk, terwyl apoptose verhoed word via die inhibering van sitochroom c lekkasie vanuit die mitochondria. Omrede grelien geen effek op die verbetering van SAD aktiwiteit getoon het in die studie nie, kan die anti-oksidant effek nie in hierdie weefsel bewys word nie. Gevolglik kan ons wel lig werp op die meganismes waarby grelien, DOX se newe-effekte kan teëwerk, maar verdere navorsing is nodig om die potensiaal van grelien as ‘n adjuvante terapie vir DOX-geïnduseerde pankreatiese skade voor te stel.

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