Browsing by Author "Adonis, Muneeb"

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    A matter of life or death : autophagy in the context of prolonged doxorubicin therapy
    (Stellenbosch : Stellenbosch University, 2019-04) Adonis, Muneeb; Sishi, Balindiwe J. N.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Introduction Doxorubicin (DOX) is an effective treatment against a variety of cancers, and thus remains a commonly used chemotherapeutic drug. The chief side effect of DOX treatment is cardiotoxicity. The precise mechanisms by which DOX induces cardiotoxicity are unknown. One of the most accepted mechanisms is the excess production of ROS. The structure of DOX allows it to undergo redox cycling and form DOX-iron complexes, both of which generate free radicals. DOX-induced oxidative damage is more prevalent in the heart than other organs. As the pathogenesis of cardiotoxicity appears to be mediated by oxidative stress, it seems as if the most effective treatment would be antioxidant therapy. Antioxidant therapy has proved unsuccessful. Autophagy is a catabolic process which allows a cell to remove cytoplasmic components. Components regarded as surplus or defective may be removed to ensure the survival of the cell. There have been numerous studies conducted to evaluate the relationship between DOX and autophagy. Many of these studies have concluded that DOX treatment does affect autophagy. Although there is literature to support both the up and downregulation of autophagy. We hypothesis that during the development of DOX-induced cardiotoxicity autophagy is downregulated. Attenuation of this downregulation during DOX therapy will attenuate the effects of cardiotoxicity. Methodology Three-week-old male black 6 (C57BL/6) mice were split into six groups; vehicle control, DOX, rapamycin, a starvation, DOX and rapamycin combination and DOX and starvation group. The DOX group received 2mg/kg DOX weekly for a total of eight weeks, resulting in a cumulative dose of 16mg/kg. The rapamycin group received 2mg/kg rapamycin weekly. The DOX and rapamycin group received rapamycin 30 minutes prior to receiving DOX. The starvation group was starved from food for 24 hours each week but were allowed free access to drinking water. The DOX and starvation group were starved for 24 hours prior to receiving DOX. A week after the final treatment, mice were euthanised. The whole hearts were harvested and sectioned, roughly into halves. One half of each was stored in formaldehyde solution for histological analysis, the other was frozen in liquid nitrogen for biochemical analysis. H&E staining was performed to assess tissue morphology and cardiomyocytes area. Picrosirius red staining was performed to assess fibrosis. Fluorometry was done to assess oxidative stress via GSH, GSSG and CDs levels. Western blots were performed to assess expression of caspase 3 and LC3. A Kolmogorov- Smirnov normality test was done to test for a normal distribution. Appropriate statistical tests were applied. P-values were considered significant when P < 0.05. Results Differences in body mass first appeared at week 5, with both DOX+ Rapa and DOX+ starve weighing less than control. Differences then appeared (at week 6) between DOX and control, with DOX weighing less than control. These differences continued until the end of the study. Histology showed cardiomyocyte area was decrease in the DOX group (97.4± 4.1) the control (112.6± 4.3). There was also an increase in fibrosis in the DOX group (2.07± 0.22) compared to the control (1.05± 0.18) and the DOX+ starve group (1.21± 0.18) had less fibrosis than DOX alone. Biochemical analysis showed autophagy was decreased in the DOX group (0.3733± 0.0683) compared to the control group (1.1420± 0.2262). However, the DOX+ Rapa (0.4344± 0,0543) and DOX+ starve (0.4818± 0.1240) groups also had decreased autophagy compared to the control group. Discussion The histological analysis confirms cardiotoxicity as DOX caused cardiac damage. Biochemical analysis was less straightforward. Oxidative stress markers were tested for too long after levels may have been detectable. While this provides no oxidative stress data, it suggests the cardiotoxicity was chronic rather than acute, as cardiac damage persisted after the stress had ended. Apoptotic data was inconclusive. DOX treatment caused a decrease in autophagy. Meaning, as hypothesised, this could be the target of an adjuvant therapy. The adjuvant treatment groups showed some improvement when compared to the DOX group. Both the DOX+ Rapa and DOX+ starve group showed histological improvement. The LC3 data however showed, the adjuvant therapies did not upregulate autophagy. Neither DOX+ Rapa nor DOX+ starve showed any increases in autophagy when compared to DOX. Due to the lack of attenuation of autophagic dysregulation, the reason for the protective effect of the adjuvant therapy is a mystery. Regardless, this study suggests that autophagic upregulation a potential adjuvant therapy.