Browsing by Author "Goldswain, Toni Leigh"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    An investigation into the potential cardioprotective effects of ghrelin in a rat model of chronic Doxorubicin-induced cardiotoxicity
    (Stellenbosch : Stellenbosch University, 2017-12) Goldswain, Toni Leigh; Sishi, Balindiwe J. N.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH SUMMARY: Introduction Doxorubicin (DOX) is an anthracycline that has significantly improved the outcome of cancer patients since its discovery. However, its clinical success remains limited due to the dose-dependent cardiotoxic side effects associated with its use. While the mechanisms responsible for this condition are still not well defined, oxidative stress alongside apoptosis, remain the classical but major contributors. Ghrelin, an endogenous brain-gut peptide, most well-known for its effects on appetite and growth hormone release, has been shown to exert anti-apoptotic, antioxidative, anti-inflammatory and anti-fibrotic effects in several models of cardiovascular disease. These cardioprotective effects offered by ghrelin have been indicated to occur through the activation of the pro-survival proteins, ERK1/2, Akt/PKB and STAT3. This study therefore investigated the protective effects of ghrelin in a chronic model of DOXinduced cardiotoxicity. Methods Male Sprague-Dawley rats were acclimatized and divided into five experimental groups. While the control group remained untreated, the ghrelin group received 300 μg/kg ghrelin per week, the DOX group received 2.5 mg/kg per week, the combination group received both DOX and ghrelin at the previously mentioned doses and the vehicle group received saline. All injections were performed via intraperitoneal injection for eight weeks. One week after the last injection, the rats were euthanized, blood was collected and the hearts were subjected to a 40-minute working heart perfusion protocol to obtain functional data. The hearts were weighed and then cut transversely into two sections, where one half was snap frozen for biochemical analysis and the other half was preserved in formalin for histological analysis. Cardiovascular markers of damage and pro-inflammatory cytokines were measured in serum using a luminex assay, and fibrosis and general morphology were assessed using the Masson’s Trichrome and H&E stains, respectively. Cytochrome c expression was evaluated by immunohistochemistry, while oxidative stress was assessed using the TBARS, conjugated diene, ORAC, SOD and glutathione assays. Apoptosis was determined using the Caspase Glo® assay and the expression of pro-survival proteins was measured using Western blot analysis. Results During the eight weeks of treatment, DOX significantly reduced weight gain and food consumption, whereas ghrelin maintained normal body weight and stimulated appetite. As expected, DOX induced significant oxidative stress when compared to the control, as demonstrated by the formation of conjugated dienes (1.64 ± 0.11 vs 0.55 ± 0.12 μmol/gram, p = 0.0003) and a reduction in the GSH:GSSG ratio (2.10 ± 0.47 vs 9.66 ± 1.08 arbitrary units, p< 0.05), whereas ghrelin attenuated these effects. Apoptotic cell death was also induced, as evident by an increase in cytochrome c staining, caspase activity (67877 ± 15686 vs 13421 ± 1871 relative light units, p< 0.0001) and PARP cleavage (2.11 ± 0.24 vs 1.00 ± 0.09 fold change, p = 0.0081). The decrease in cell death and oxidative stress in this scenario was associated with a reduction in TNF- and an improvement in cardiac function, which was otherwise worsened in the DOX group due to the decline cardiac output, coronary flow, ratepressure product, left ventricular developed pressure and total work. Even though ghrelin treatment in the presence of DOX did not induce noteworthy changes in the protein expression of ERK1/2 and Akt/PKB, the phosphorylation of STAT3 was improved with ghrelin administration. Discussion and Conclusion The observations presented in this study indicate that while DOX is a known effective chemotherapeutic agent, it produces cardiotoxic effects through the induction of oxidative stress and consequently apoptosis, possibly due to the downregulation of ERK1/2 and Akt/PKB. The co-administration of ghrelin with DOX significantly decreased the detrimental effects associated with DOX treatment alone. The effects of ghrelin were not only beneficial at organ level, but also at the organism level, as a result of improved general well-being of the experimental animals and through the maintenance of cardiac function, a decline in myocardial TNF- production and the stimulation of the STAT3 signaling pathway. The fact that ghrelin alone did not exert any detrimental effects makes this peptide an appealing cardioprotective agent and may therefore have the potential to improve the high morbidity and mortality rates of cancer survivors. While ghrelin in this context may possess anticardiotoxic effects, further research is required to determine the effects of ghrelin on cancer cell proliferation.
  • Thumbnail Image
    Item
    The (un)SAFE and RISK(y) sides of doxorubicin-induced cardiotoxicity
    (Stellenbosch : Stellenbosch University, 2014-12) Goldswain, Toni Leigh; Sishi, Balindiwe J. N.; Lacerda, Lydia; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Introduction The discovery of Doxorubicin in the 1960s has drastically improved the survival rates of cancer patients, however, its success is limited by dose-dependent cardiotoxicity. While much of the literature has focused on acute cardiotoxicity which is minor and generally reversible, chronic cardiotoxicity poses a serious threat to cancer survivors since it can lead to dilative cardiomyopathy, congestive heart failure and even death. The mechanisms that contribute to cardiotoxicity are still a matter of controversy, however, oxidative stress-induced myocardial damage and apoptosis are thought to be the major role players. Reperfusion injury, also characterized by oxidative stress and apoptosis, occurs as a result of restoring blood flow to an ischemic heart. Fortunately, pre- and post-conditioning are techniques employed to minimize this damage and are thought to do so by activating the reperfusion injury salvage kinase (RISK) and survivor activating factor enhancement (SAFE) pathways. The RISK pathway involves the pro-survival kinases, Erk1/2 and Akt, while the SAFE pathway, triggered by TNF-α, involves Jak2 and STAT3. Since both reperfusion injury and Doxorubicin-induced cardiotoxicity share similar characteristics, this study aimed to determine whether the RISK and SAFE pathways are activated in response to long-term Doxorubicin treatment. Furthermore, this study aimed to determine whether TNF-α is produced during treatment, since its role in Doxorubicin-induced cardiotoxicity is still relatively unknown. Methods H9c2 cardiomyocytes and differentiated C2C12 myotubes were treated daily with increasing concentrations of Doxorubicin for a total of 120 hours. Cell viability, apoptosis and necrosis were assessed using the MTT, Caspase-Glo® 3/7 and lactate dehydrogenase assays respectively. TNF-α production was measured using Quantikine® ELISA kits and various assays were used to assess oxidative stress, anti-oxidant capacity and anti-oxidant status. The protein expression of the RISK and SAFE pathways were analysed by western blotting using both phospho-specific and total antibodies. Results and Discussion Treatment with Doxorubicin caused a time- and dose-dependent decrease in cell viability in both cell lines and this was accompanied by an increase in apoptosis. In the H9c2 cardiomyocytes, treatment with 0.2 μM Doxorubicin yielded significant levels of TNF-α after 120 hours and we can speculate that these low levels partially protected the cells from the toxic effects of Doxorubicin by activating the SAFE pathway, since both Jak2 and STAT3 were phosphorylated at this concentration. Treatment with 1 μM Doxorubicin caused a larger and biphasic pattern of TNF-α release, which may have then contributed to the decrease in cell viability, since the SAFE pathway was not activated at this concentration. Akt was phosphorylated during the first 72 hours of treatment with the low dose of Doxorubicin, but chronic treatment prevented this phosphorylation. While Erk1/2 was not phosphorylated at all at the low dose of Doxorubicin, neither Akt nor Erk1/2 was phosphorylated at the high dose and their inhibition may contribute to the cardiotoxic effects of Doxorubicin. In the C2C12 myotubes, a significant amount of TNF-α was produced after 120 hours of treatment with the low dose of Doxorubicin. Treatment with the high dose of Doxorubicin induced significant TNF-α production at every time point. While STAT3 was phosphorylated at the serine residue after treatment with the low dose of Doxorubicin, treatment with the high dose induced phosphorylation at the tyrosine residue in a time-dependent manner. p-Jak2 expression was significantly down-regulated at both concentrations of Doxorubicin, suggesting that STAT3 proteins can by-pass activation by Jak2. The Erk1/2 leg of the RISK pathway was also not activated for the majority of the treatment period, however, p-Akt expression was increased at the low concentration of Doxorubicin relative to total Akt expression. Conclusion These observations indicate that treatment with Doxorubicin causes a severe, dose-dependent loss in viability which is likely to mediated by high concentrations of TNF-α (induced by high concentrations of Doxorubicin) and down-regulation of protective signaling pathways. TNF-α may confer partial protection at low concentrations by activating the SAFE pathway. However, activation of the SAFE pathway could not provide sufficient protection from Doxorubicin, most probably because the RISK pathway was not simultaneously activated. Our results also clearly highlight the differences between acute and chronic treatment since a single high dose of Doxorubicin produced vastly different responses to cumulative treatment with a low dose. Before one can extrapolate these results into the clinical setting, further research is required to provide a better understanding of the RISK and SAFE pathways and whether stimulation thereof will provide a protective effect. In addition, although our study has shown that TNF-α is produced in response to Doxorubicin treatment, its true role, whether beneficial or detrimental, remains to be determined.