Masters Degrees (Physiological Sciences)

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Acute simulated hypoxia and ischemia in cultured C2C12 myotubes : decreased phosphatidylinositol 3-kinase (PI3K)/Akt activity and its consequences for cell survival
(Stellenbosch : Stellenbosch University, 2008-12) Thomas, Mark Peter; Engelbrecht, Anna-Mart; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
Cells are equipped with an array of adaptive mechanisms to contest the undesirable effects of ischemia and the associated hypoxia. Indeed, many studies have suggested that there is an increase in the PI3K/Akt pathway activation during hypoxia and ischemia. Damaged muscle can be regenerated by recruiting myogenic satellite cells which undergo differentiation and ultimately lead to the regeneration of myofibres. The C2C12 murine myogenic cell line is popular for studying myogenesis in vitro, and has been used in many studies of ischemic microenvironments. PI3K/Akt pathway activity is increased during C2C12 myogenesis and this is known to produce an apoptosis resistant phenotype. In this study, we provide evidence that high basal levels of PI3K activity exist in C2C12 myotubes on day ten post-differentiation. Ischemia is characterized by depleted oxygen and other vital nutrients, and ischemic cell death is believed to be associated with an increasingly harsh environment where pH levels decrease and potassium levels increase. By employing a model that mimics these changes in skeletal muscle culture, we show that both acute simulated ischemia and acute hypoxia cause decreases in endogenous levels of the p85 and p110 subunits of PI3K and a consequent reduction in PI3K activity. Supplementing skeletal muscle cultures with inhibitors of the PI3K pathway provides evidence that the protective effect of PI3K/Akt is subsequently lost in these conditions. Using Western blot analysis, a PI3K ELISA assay as well as known inhibitors of the PI3K pathway in conjunction with the MTT assay we are able to demonstrate that the activation of downstream effectors of PI3K, including Akt, are concurrently decreased during acute simulated ischemia and acute hypoxia in a manner that is independent of PDK-1 and PTEN and that the decreases in the PI3K/Akt pathway activity produce a knock-on effect to the downstream signalling of transcription factors, such as Fox01 and Fox04, in our model. We proceed to provide compelling evidence that the apoptotic resistance of C2C12s is at least partially lost due to these decreases in PI3K/Akt pathway activity, by showing increased caspase-3 and PARP cleavage. Then, using vital staining techniques and a DNA fragmentation assay, we demonstrate increased cell membrane impairment, cell death and apoptosis after three hours of simulated ischemia and hypoxia in cultured C2C12 myotubes. In addition to the main findings, we produce evidence of decreased flux through the mTOR pathway, by showing decreased Akt-dependant phosphorylation at the level of TSC2 and mTOR during simulated ischemia and hypoxia. Finally, we present preliminary findings indicating increased levels of HIF1α and REDD-1, representing a possible oxygen sensing mechanism in our model. Therefore, we show that there is in fact a rapid decrease in PI3K/Akt activity during severe, acute simulated ischemia and hypoxia in C2C12 myotubes on day ten post-differentiation, and this causes a concomitant down regulation in cell survival pathways and increased activity of cell death machinery. Thereafter, we propose a possible mechanism of action and provide a platform for future studies.
Ageing-associated oxidative stress and inflammation are alleviated by products from grapes
(Hindawi, 2016) Petersen, K. S.; Smith, Carine
Advanced age is associated with increased incidence of a variety of chronic disease states which share oxidative stress and inflammation as causative role players. Furthermore, data point to a role for both cumulative oxidative stress and low grade inflammation in the normal ageing process, independently of disease. Therefore, arguably the best route with which to address premature ageing, aswell as age-associated diseases such as diabetes, cardiovascular disease, and dementia, is preventative medicine aimed at modulation of these two responses, which are intricately interlinked. In this review, we provide a detailed account of the literature on the communication of these systems in the context of ageing, but with inclusion of relevant data obtained in other models. In doing so, we attempted to more clearly elucidate or identify the most probable cellular or molecular targets for preventative intervention. In addition, given the absence of a clear pharmaceutical solution in this context, together with the everincreasing consumer bias for natural medicine, we provide an overview of the literature on grape (Vitis vinifera) derived products, for which beneficial effects are consistently reported in the context of both oxidative stress and inflammation.
An alternative approach to premature luteal regression
(Stellenbosch : Stellenbosch University, 2006-12) Pretorius, Willem S.; Barry, Daniel Malan; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Premature luteal regression occurs on average in 30% of superovulated sheep ewes. This phenomenon occurs early in the cycle before the embryo’s can be collected and is a major contributor to failure in embryo transfer programs. This research was done to understand the physiological mechanisms involved. Chapter two provides a general background of the physiology of natural luteolysis and the maternal recognition of pregnancy. The chapter introduces some new concepts on the topic of cell death and provides a recent literature review on research done on the phenomenon of premature luteal regression. This chapter forms the base of ideas and arguments that follows in the two studies containing new original work in this field. The research contained in this thesis comprises of two in vivo studies. The first study (Chapter 3) compare premature luteal regression to Prostaglandin F2α (PGF2α) induced regression with emphasis on the changes in levels of the steroid hormones progesterone (P4) and estradiol - 17β (E2-17β) and changes in structure and ultra structure. The following conclusions were made: 1. Premature luteal regression is not merely inadequate luteal support, but indeed early luteal regression, since seasonal influences could merely be nutritional influences, and a definitive increase in P4 were recorded in animals exhibiting the phenomena. 2. Nutritional influences could play a role, but the type and quality of nutrients and mechanism involved, is still unclear. 3. PGF2α-induced regression differs from premature luteal regression in that: a) The progression of functional and structural regression in PGF2α -induced regression is slower than in premature luteal regression. b) Regressed corpora lutea do not occur with normal functioning corpora lutea. 4. There is a distinct second E2-17β peak preceding the decline in P4 in animals that exhibits signs of premature luteal regression. A threshold initiating premature luteal regression was not established. The second study (Chapter 4) compares the changes in the ovine β estradiol - 17 β receptor (oERβ) between premature luteal regression and PGF2α induced regression. The study concludes that there could be a potential role for oERβ in premature luteal regression. The findings of these two studies raise some questions about the conventional perception that early release of PGF2α is the cause of premature luteal regression. The thesis concludes in a hypothesis (Chapter 4) explaining the phenomenon.
Anthropometric characteristics and changes with HIV and ART in a randomly selected population in the Drakenstein region Western Cape Province
(Stellenbosch : Stellenbosch University, 2014-12) Beukes, Dillan Charles; Nell, Theo A.; Essop, M. Faadiel; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Background - Highly active antiretroviral therapy (HAART) has extended life expectancy and enhanced the well-being of HIVpositive individuals. Since there are concerns regarding HAART-mediated onset of cardio-metabolic diseases in the long-term, we evaluated the anthropometric profile of HIV-infected individuals in the Drakenstein District (Western Cape, South Africa). - Objective of study - The primary objective of this study was to document the anthropometric characteristics within and HIV infected population in the Drakenstein region of the Western Cape Province of South Africa. - Methods - HIV-positive patients (n=44 males, n=102 females; 20-40 yrs.) were recruited for three groups: 1) control (HIVnaïve), 2) HIV-positive (HAART ≤ 0-36 months), and 3) HIV-positive (HAART ≥ 36 months). Participants underwent a) anthropometric (triceps skin fold [TSF], and b) bioelectrical impedance measurements (body cell mass [BCM], fat free mass [FFM], protein, muscle mass (MM), mineral, total body potassium (TBK) and calcium (TBCa), glycogen, and fat mass [FM]). - Results - Our data reveal that HIV-positive males on HAART ≤ 0-36 months displayed a trend for lower body cell mass (BCM), fat free mass (FFM), fat mass (FM), triceps skinfold (TSF) and protein content (vs. control). Females exhibited reduced BCM (p=0.001) and lower protein (p=0.003), muscle mass (p=0.001), glycogen (p=0.001), FM (p=0.0005) and FFM (p=0.002) content. However, with longer-term treatment (HAART ≥ 36 months), females displayed higher BCM (p=0.0001), protein (p=0.01), muscle mass (p=0.0003), glycogen (p=0.0001), FM (p=0.00003) and FFM (p=0.0002) vs. the 0-36 months treatment group. Their waist-to-hip ratio also increased vs. the naïve female group (p=0.02). By contrast, males on HAART ≥ 36 months did not show any significant increases vs. the HAART ≤ 0-36 month’s group. - Conclusions - This study demonstrates observed striking gender-based anthropometric differences in South African HIVpositive individuals on HAART. While both genders initially exhibit muscle wasting, HIV-positive females show a strong improvement with longer-term treatment vs. males. However, higher abdominal fat accumulation in females with longer-term treatment potentially increases their risk for the future onset of cardio-metabolic complications.
Antioxidant (Oxiprovin TM) supplementation and muscle recovery from contusion injury - an in vivo study
(Stellenbosch : Stellenbosch University, 2007-12) Kruger, Maria Jacoba; Smith, Carine; Smith, R. M.; Myburgh, Kathryn H.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Human studies on the response of muscle to contusion injury are limited, probably due to the large variability in injury severity and the non-specificity of clinical symptoms reported. To circumvent this problem, several experimental animal models have been designed to study muscle damage and regeneration after contusion injuries. However, the majority of techniques currently used to induce contusion injury are very invasive and therefore not optimal. Furthermore, published studies regarding clinical treatment of such injuries are limited. The main aims of this study were therefore: a) to establish and characterise an in vivo model of non-invasive contusion injury, and b) to assess the effect of pre-injury chronic administration of the antioxidant supplement Oxiprovin™ - a natural grape seed extract (GSE) - on skeletal muscle recovery after experimentallyinduced injury. Two groups of male Wistar rats were subjected to 14 days of oral administration of isovolaemic placebo (sterile isotonic saline) or GSE (20 mg/kg/day) prior to induced contusion. Contusion injury was induced with the mass-drop technique, and recovery parameters assessed for up to 14 days post-injury. Placebotreated rats on average exhibited a 56 % higher creatine kinase (CK) activity when compared to the GSE-treated rats when area under the curve (AUC) was calculated for 14 days post-injury (p < 0.001). In the placebo group, plasma oxygen radical absorbance capacity (ORAC) was unchanged over time, but muscle ORAC was significantly increased by day 7 post-injury (p < 0.001). In the GSE group, a significant decrease in both plasma (p < 0.01) and muscle ORAC (p < 0.001) was evident 4 hr after injury, followed by a significant increase by day 3 (p < 0.05 and p < 0.001 respectively). CD34+ satellite cell (SC) numbers (quiescent and activated) peaked earlier in GSE-treated rats when compared to placebo-treated rats (4 hours vs. day 7 post-injury). Total satellite cell number (CD56+) also peaked earlier in GSE-treated rats than in placebo-treated rats (4 hours vs. 3 days post-injury), while M-cadherin+ SC numbers (quiescent, activated or proliferating) in both treatment groups were significantly increased 4 hours post-injury (p < 0.001), but more so in the placebo group. In GSE-treated rats when compared to placebo-treated rats, newly generated muscle fibres (displaying central nuclei and MHCf +) both appeared (day 3 vs. day 7 post-injury) and peaked in number (day 3 vs. day 7 post-injury; increase from baseline p < 0.001 for both) earlier. The results of this study demonstrate that we have successfully established an in vivo model for non-invasive contusion injury in rats. Furthermore, we have shown that Oxiprovin™: a) increased the ability to scavenge reactive species generated after injury and b) resulted in the activation of satellite cells and formation of newly generated muscle fibres at an earlier time point, thus accelerating the recovery of skeletal muscle after a standardised contusion injury.
Are early proteomic and metabolic changes induced by long-term sugar-sweetened beverage consumption the key to unlocking the cardio-metabolic pandemic?
(Stellenbosch : Stellenbosch University, 2017-03) Benade, Janina; Essop, M. Faadiel; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: INTRODUCTION: Cardio-metabolic diseases (e.g. type 2 diabetes mellitus) are a major cause of mortality worldwide. The incidence of cardio-metabolic diseases continues to increase, especially in low and middle income countries. This “pandemic” is possibly brought about by a fairly universal shift towards a more “Westernized” diet. High sugar consumption - a hallmark of the “Westernized” diet - may play a key role in the onset of cardio-metabolic diseases. Accordingly, our research focus moved towards sugar-sweetened beverages (SSBs) as it is a major source of added dietary sugars. The current study aimed to elucidate underlying mechanisms leading to the development of cardiometabolic diseases by exploiting a novel rat model of long-term SSB intake, and by focusing on the liver as a major metabolic organ. Here we evaluated well-known systemic markers together with hepatic proteome analysis and downstream consequences. METHODS: Male Wistar rats ( 200 g) were gavaged with 3-5.1 mL SSB daily for three and six months, respectively. The two control groups were gavaged with an iso-volumetric amount of water and iso-caloric amount of butter, respectively. Body weight and systemic blood markers were measured. A proteomic expression analysis was performed on the six-month liver samples. The rest of our experimental work was guided by the proteomic results. Four markers for oxidative stress were evaluated: malondialdehyde, conjugated dienes, reduced:oxidized glutathioneand oxygen radical absorbance capacity. The non-oxidative glucose pathways (NOGPs): polyol pathway, hexosamine biosynthetic pathway, advanced glycation end-products formation and protein kinase C activation; were measured as elevated activity could be indicative of impaired glycolytic flux. The liver histology was investigated with Hematoxylin and Eosin and Masson’s Trichrome stains, respectively. Finally, Western blotting techniques were used to evaluate markers of inflammation. RESULTS: SSB consumption had little effect on systemic markers of cardio-metabolic health. Our proteomic analysis revealed that the expression level of 140 proteins was significantly altered in the SSB group, with a major finding that SSB consumption induces hepatic endoplasmic reticulum (ER) stress. Initially the liver adapted to SSB-mediated nutrient overload by increasing oxidative phosphorylation, suppressing protein transcription, degrading misfolded proteins and improving protein folding capacity. However, due to prolonged stress liver cells entered an ‘’alarm phase’’ marked by a decrease in mitocholdrial metabolism. The proteomic results further revealed that SSB-induced effects are largely attributed to excess caloric intake versus SSBs per se. Surprisingly, oxidative stress did not precede ER stress as there were no significant changes in any of the oxidative stress markers here evaluated. The activity of the NOGPs did not increase significantly thus suggesting that moderate SSB intake did not suppress glucose metabolism and the glycolytic pathway in particular. Conversely, SSB intake increased hepatic lipid storage while limited changes were detected between the groups regarding inflammation and stress signaling. CONCLUSION: Frequent SSB consumption triggers metabolic changes in the liver, i.e. ER stress despite the lack of obvious manifestation of macroscopic “warning signs”. Thus the current study identifies hepatic ER stress as a relatively early result of long-term SSB consumption and it therefore emerges as a unique therapeutic target.
ART-related body composition changes in adult women in a semi-rural South African context
(Stellenbosch : Stellenbosch University, 2006-12) De Bruto, Petro C.; Myburgh, Kathryn H.; Smith, Carine; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: The aim of this study was to investigate practical methods of monitoring AIDS related wasting and lipodystrophy in a resource-poor clinical setting with HIV infected women as the population group of interest. Measurement of body composition changes using anthropometry is both cost- and time-efficient. Various different skinfolds were taken and two different equations (the equations of Pollock et al. (1975) and Durnin and Womersley (1974) for calculating body fat were used to determine the most promising method or methods of monitoring body composition changes in a clinical setting. Detailed anthropometric measurements were performed, as well as selected measurements for haematological parameters and quality of life (QoL) for a group of 8 participants on antiretroviral medication (ART group) and 6 participants who were not on treatment (TN group). New variables namely, intra-abdominal indicator (IAI) and a percent of ideal body mass to percent of ideal arm circumference ratio (%IBW:%IAC) were investigated as possible indicators of lipodystrophy. Although measurements were taken at various timepoints, three specific time-points were chosen for data-analysis for the ART group and two time points for the TN group. These three time-points were, baseline (on the day of recruitment for TN participants and within one month before the initiation of treatment for ART participants), short-term (2 to 12 weeks after treatment initiation or the baseline measurement or for the ART and the TN participants) and long-term (within one and a half year of treatment initiation for the ART group). ART and TN participants did not differ for many variables at baseline. The major differences between ART and TN were in measured and derived variables of the arm, especially percent of ideal arm circumference (%IAC) and upper arm fat area (UAFA), which were significantly lower in the ART group. CD4+ and QoL improved significantly for the ART participants from baseline to long-term. This was not associated with changes in muscle mass, but rather some fat mass variables. Participants on antiretroviral medication exhibited changes relating to abdominal obesity. It was concluded that antiretroviral therapy contributed greatly to the QoL of the participants and it probably aided in the recovery from wasting for at least one participant in this study. Measures of the arm can be used in a rural clinical setting to effectively monitor patients with regard to AIDS related wasting. The new variables IAI and %IBW:%IAC could be helpful in the monitoring of lipodystrophy and should be investigated in future research.
An assessment of ischemia-reperfusion injury in rats exposed to chronic psychological stress
(Stellenbosch : Stellenbosch University, 2019-12) Oliver, Lukas Van Zyl; Essop, M. Faadiel; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Cardiovascular disease remains the leading cause of death worldwide. Apart from known risk factors such as poor dietary intake, physical inactivity and smoking, chronic psychological stress is emerging as an important modifiable risk factor in the development of cardiovascular disease. The body relies on two physiological mechanisms to counter acute stressors and to achieve and/or maintain homeostasis, i.e. the hypothalamic-pituitary-adrenal axis and the autonomic nervous system. However, chronic activation of these systems can lead to the disruption of cellular and systemic processes that could potentially result in the development of neurological or psychosomatic diseases. Both chronic stress and ischemia-reperfusion injury are associated with a robust inflammatory response and the induction of oxidative stress. Does chronic psychological stress render the heart more susceptible to ischemia and reperfusion damage, and what are the role(s) of oxidative stress and inflammation in stress-related cardiac dysfunction? These questions will form the basis of this review. Following a comprehensive review, we established that chronic stress does render the heart more susceptible to damage following ischemia-reperfusion. After reviewing the mechanisms involved in both ischemia-reperfusion and chronic stress, we hypothesized that chronic stress induced inflammation and oxidative stress are major contributors in aggravated ischemia-reperfusion injury.
Association between antioxidant status and MnSOD Ala-9Val polymorphism in trained male athletes (rugby players) and sedentary male students controlled for antioxidant intake
(Stellenbosch : Stellenbosch University, 2007-03) Seele, Maria; Senekal, M.; Steyn, N. P.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: The human body has developed an integrated antioxidant defence system to protect against free radical damage. Acute exercise may result in the increased generation of free radicals, including reactive oxygen species, and this may overwhelm antioxidant defence systems resulting in oxidative stress. However, it has been shown that individuals who undergo regular exercise training may have improved antioxidant capacity when compared to sedentary controls. Results from research regarding the association between antioxidant capacity and exercise training are however not conclusive and further investigation is required. Therefore, the aim of this study was to investigate the association between the total plasma antioxidant status and selected plasma indicators of antioxidant status and the MnSOD Ala-9Val (-28C®T) polymorphism in trained male athletes (rugby players) and sedentary male students while controlling for dietary intake of the major antioxidants using a validated dietary assessment method. In order to address the potential confounding effect of dietary antioxidant intake on antioxidant status in the main study, a FFQ that measures vitamin C, vitamin E, carotenoid and flavonoid intake was developed. The reproducibility was assessed by the repeat administration of the FFQ (n = 38), while the va lidity was assessed using a 28-day closeended dietary record and repeated plasma vitamin C values (n = 18). Several statistical tests were conducted to compare the values obtained from the FFQ with values obtained from the various reference methods. While results from Bland-Altman plots suggested that the reproducibility and validity of FFQ was not completely satisfactory, similar mean values, moderate to strong correlation coefficients, and a high percentage of individuals classified correctly according to quartiles of intake indicated satisfactory reproducibility and validity of the FFQ in assessing antioxidant intake. Furthermore, moderate to strong validity coefficients obtained from the method of triads also indicated satisfactory validity for the FFQ. The main study involved a cross-sectional study that compared plasma vitamin C and carotenoid levels as well as total plasma antioxidant status in trained rugby players (n = 76) and sedentary male subjects (n = 39) with different MnSOD genotypes, while controlling for dietary antioxidant intake. Rugby players had significantly higher plasma vitamin C and carotenoid levels compared to sedentary students, which indicated more satisfactory plasma antioxidant status. This was also reflected in the tendency for total plasma antioxidant status (ORAC assay) to be higher in rugby players than sedentary students. MnSOD genotype did not influence plasma vitamin C and carotenoid levels or plasma total antioxidant status, with or without control for dietary antioxidant intake. Dietary vitamin C, vitamin E, carotenoid an flavonoid intake (from foods + supplements) was similar for rugby players and sedentary students and was adequate for both groups. Thus the association between antioxidant status and MnSOD genotype in rugby players and sedentary students seemed not to be influenced by dietary antioxidant intake. In conclusion therefore, rugby players undergoing regular exercise training had a more satisfactory antioxidant status compared to sedentary students. Based on this conclusion, the widespread use of antioxidant supplements by athletes is questioned.
Association between cancer, adipose tissue and selected systemic markers : a possible classification according to body shape
(Stellenbosch : Stellenbosch University, 2016-03) Mentoor, Ilze Lauren; Nell, Theo A.; Kruger, Maritza J.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Background: The metabolic syndrome (MetS) is a cluster of risk factors associated with an increased risk of developing chronic diseases of lifestyle, and has more recently been associated with cancer risk. Currently, the pathophysiology of the MetS and cancer risk is still unknown; however it is proposed to involve several factors. These include the effects of body composition (android and gynoid shapes), and insulin resistance on the bioavailability of growth factors, inflammatory markers and sex hormone profiles. Various anthropometrical measurements have been used to investigate body composition, however, due to their limitations, a new metric namely a body shape index (ABSI) has been proposed to be a better measure of fat distribution and body shape. Aims: To determine the prevalence of the MetS, and the possible risks of developing cancer in relation to metabolic status, body composition, growth factors as well as inflammatory and sex hormone parameters. Methods: Female participants between the ages of 20-60 years were classified according to the International Diabetes Federation’s (IDF) definition of the MetS and according to body shape (android/gynoid) by photoscopic somatotyping. A series of tests and assessments were conducted; such as blood pressure assessments, anthropometric measurements, bioelectrical impedance analyses (BIA) and blood analyses. Blood analysis included fasting glucose, fasting insulin, lipid profile, insulin-like growth factor-1 (IGF-1), inflammatory marker (C-reactive protein (CRP)); and sex hormone parameters (oestrogen, female testosterone, sex hormone binding globulin; and free androgen index). Results: The prevalence of the MetS was found to be 57.5 %; with abdominal obesity (73.8 %), elevated blood pressure (BP, 68.8 %) and low high density lipoprotein-cholesterol (HDL-c) levels (68.8 %) being the more prevalent risk factors. Both metabolic status; and body shape alone were found to be predictors influencing anthropometric, BIA, physiological and biochemical blood parameters. Metabolic status was found to have an effect on several parameters in the gynoid body shape groups, i.e. body mass (BM) (p<0.001), hip circumference (HC) (p<0.01), body mass index (BMI) (p<0.001), fat mass (FM) (%) (p<0.01), fat free mass (FFM) (%) (p<0.01), waist circumference (WC) (p<0.001), HDL-c (p<0.001), triglycerides (TG) (p<0.05), systolic blood pressure (SBP) (p<0.05) and diastolic blood pressure (DBP) (p<0.01), while metabolic status showed an effect on BM (p<0.001), BMI (p<0.01), TG (p<0.05), SBP (p<0.01) and DBP (p<0.01) in the android body shape groups. Both metabolic status and body shape did not show any effect on ABSI, total cholesterol (TC), low density lipoprotein-cholesterol (LDL-c), fasting insulin, CRP and all sex hormone parameters. Correlation analyses revealed significant correlations for several anthropometric, BIA and blood parameters. Conclusion: This study showed that metabolic status, body shape and/or both could predict changes in various body composition, physiological and biochemical parameters in women. However, no effects were evident for any parameters linking the MetS to cancer risk. Thus, no accurate conclusion could be drawn regarding the pathophysiology. Our findings on ABSI, still warrants future investigation to substantiate the use of this metric in relation to the MetS, body shape and cancer risk.
The association between the metabolic syndrome and bone mineral density in pre- and post-menopausal farm workers
(Stellenbosch : Stellenbosch University, 2016-12) Marais, Sumine; Nell, Theo A.; Kruger, Maritza J.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Introduction and aims: The prevalence of the metabolic syndrome (MetS) is increasing, both globally and in South Africa. Albeit so, limited data have been published in the South African context. One of the factors that appear to influence the prevalence of the MetS is menopausal status, with both the MetS, and menopausal status influencing bone mineral density (BMD); however, the reported results are inconsistent. Therefore, the aim of this study was to determine the prevalence of the metabolic syndrome, investigating bone health as well as the interactions between the MetS, menopausal status and bone health, in a farm working female population in the Western Cape. Methods: A total of n=80 females were recruited and classified with the MetS, using the International Diabetes Federations’ definition. The data collected included basic anthropometric measurements, blood pressure, BMD, and several questionnaires to obtain information regarding physical activity, demographic information, menstrual-, diet- and family health- history. The blood parameters that were measured included alkaline phosphatase (ALP), vitamin D, parathyroid hormone (PTH), oestradiol (E2), fasting insulin (FI), fasting glucose (FG) and a lipid profile. Results: A relatively high prevalence of the MetS (55.0%) was reported in the current study. When investigating the separate MetS risk factors, most of the study participants had three risk factors (32.5%), with increased BP being the most prevalent MetS risk factor (72.5%). Factors that differed between MetS and Non-MetS sub-groups (according to menopausal status and age) included waist circumference (WC), high-density lipoprotein-cholesterol (HDL-c), systolic blood pressure (SBP) and diastolic blood pressure (DBP). Significant associations between body mass (BM) and E2, and body mass index (BMI) and E2, were limited to the PreM (20-39 years) age group with the MetS (r=0.58, p=0.03, and r=0.60, p=0.02). A total of 78.8% of the study participant had normal BMD. When correlating BM and speed of sound (SOS), significant associations were limited to the PreM (≥40 years) group (MetS: r=0.56, p=0.04, Non-MetS: r=0.76, p=0.00), and significant associations between BMI and SOS were noted in both PreM groups (MetS PreM 20-39 years: r=0.53, p=0.05, Non-MetS PreM ≥40 years: r=0.73, p=0.00). The significant correlations between FI and ALP (r=0.72, p=0.00), FG and ALP (r=0.89, p=0.00), and triglycerides with ALP (r=0.82, p=0.00) were limited to the PreM (≥40 years) group. Conclusion: The prevalence of the MetS was higher than that reported by previous South African studies. Irrespective of metabolic and menopausal status, most of the participants of the current study population had normal BMD. Key words: Metabolic syndrome, bone mineral density, menopause
Association between the metabolic syndrome and cancer risk : the potential role of fatty acids on body composition
(Stellenbosch : Stellenbosch University, 2016-12) Johnson, Olga; Nell, Theo A.; Kruger, Maritza; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Background: Sub-Sahara Africa is experiencing an epidemiological transition with an increasing burden of non-communicable diseases (NCDs) including obesity, hypertension, insulin resistance (IR) and dyslipidaemia. These NCD are collectively labelled the metabolic syndrome (MetS). The MetS is characterised by dyslipidaemia, and in particular, distorted fatty acid (FA) metabolism. Additionally, the MetS and its components are also associated with different FA classes. Furthermore, several lifestyle-cancers have also been associated with the MetS and its components. Aim: To determine the association and interaction between the MetS and cancer risk and the likely influence of FAs on body composition in a female population residing in the Western Cape, South Africa. Methods: Female farm workers in the Cape Winelands region (n=80) aged 20-60 years were randomly selected and categorised as having the MetS (n=34 MetS and n=46 non-MetS) using the International Diabetes Federation (IDF) criteria. All participants were additionally classified according to their body mass index (BMI). Blood pressure was measured, followed by blood sampling to determine blood glucose and insulin levels, as well as a full lipid profile. Selected red blood cell (RBC) membrane FAs and FA ratios were analysed, and enzyme-linked immunosorbent assays (ELISAs) were used to quantify serum insulin-like growth factor-1 (IGF-1) and leptin concentrations. Anthropometric measurements and bioelectric impedance analyses (BIA) were also performed. Results: The prevalence of the MetS was 42.5 % with abdominal obesity (100.0 % for the MetS, and 39.1 % for the non-MetS), hypertension (82.4 % for the MetS, and 47.8 % for the non-MetS), and low high-density lipoprotein cholesterol (HDL-c) (76.5 % for the MetS, and 34.8 % for the non-MetS) being the most prevalent MetS risk factors. Several statistically significant differences were observed between the MetS and non-MetS groups for blood parameters, including insulin and HDL-c levels (p<0.001), and glucose, IGF-1, and leptin levels (p<0.05). The MetS group also presented with significantly higher anthropometric measurements, including BMI (p<0.05), waist circumference (WC), waist-to-hip ratio (W:H), and the sagittal abdominal diameter (SAD) (all p<0.001). Furthermore, BIA (including visceral adipose tissue (VAT) area, percentage VAT (VAT %) and -subcutaneous adipose tissue (SAT %), and VAT to SAT ratio (VAT:SAT) (p<0.001 for all) also differed between the MetS and non-MetS groups. No significant differences were noted for any of the individual FAs or FA ratios. Categorisation according to metabolic status and BMI was shown to influence several metabolic-associated blood parameters, anthropometric measurements, BIA. However, metabolic status and BMI did not influence individual FA levels or FA ratios. The obese MetS group presented with significantly higher IGF-1 levels compared to their normal weight non-MetS counterparts. Correlation analyses indicated several significant associations between anthropometric measurements, BIA, FAs and metabolic-associated blood parameters. Conclusion: The results from this study suggest that metabolic status alone, and the combined effect of metabolic status and BMI, may predict alterations in metabolic-associated blood parameters, anthropometric measurements, and BIA in women, possibly linking obesity and the MetS to an increased risk of developing lifestyle-associated cancer. Keywords Metabolic syndrome, fatty acid profile, body composition, leptin, cancer risk
Can the Sutherlandia herb or resistance exercise reverse the stress inducing effects of a mild-intermittent stress procedure
(Stellenbosch : University of Stellenbosch, 2006-03) Neethling, Ian Garth; Myburgh, Kathryn H.; Smith, Carine; University of Stellenbosch. Faculty of Science. Dept. of Physiological Sciences.
This study aimed to assess the effect of mild psychological stress in male Wistar rats using incremental, intermittent stress on parameters of atrophy, including body mass, soleus and extensor digitorum longus (EDL) muscle mass, and mechanisms possibly contributing to atrophy. Serum corticosterone concentrations, 20s proteasome activity, glutamine synthetase (GS) and tyrosine amino-transferase (TAT) activities were determined. I also assessed whether Sutherlandia (Su) or resistance exercise was able to reverse the effects of stress on any of these parameters.
Cardio-metabolic effects of anti-retroviral treatment in the Cape Winelands region of South Africa
(Stellenbosch : Stellenbosch University, 2016-03) Abaid, Faten E. Bashir; Essop, M. Faadiel; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Background: Although highly active antiretroviral therapy (HAART) has significantly improved the survival of human immunodeficiency virus (HIV)-infected patients there are increased concerns regarding the onset of co-morbidities (e.g. cardio-metabolic complications) and mortalities. Although South Africa is burdened with the highest number of HIV-infected individuals globally, there is a relative paucity of data regarding potential links between HIV infection, HAART and cardio-metabolic risk/onset. Methods: This cross-sectional study therefore investigated the prevalence of cardio-metabolic risk factors in HIV-infected individuals within the Cape Winelands region of South Africa. Here we collected anthropometric, biochemical and lifestyle-related data for HIV-positive HAART naive (n =25) and HIV-positive individuals on HAART (n = 50) patients (20–55 years old) at the Worcester Community Day Centre (CDC) (Worcester, Western Cape, South Africa) during 2014 and 2015. Subjects on HAART were further divided into two sub-groupings, i.e. first line (n = 25) and second line treatments (n = 25). Results: Our data reveal the relatively high prevalence of traditional, cardio-metabolic lifestyle risk factors in HIV-infected individuals. There was a relatively high prevalence of smoking, i.e. 88% for the HIV-positive HAART naive group and 27% for the HIV-positive group on HAART (P = 0.001), while more than half of the HIV-positive individuals exhibited a positive history of familial cardiovascular diseases (CVD). There were no significant differences for fasting blood glucose (FBG) and insulin levels between HIV-positive HAART naive and HIV-positive on HAART. Lipid metabolite analyses (Total cholesterol [TC], low-density lipoprotein [LDL], high density lipoprotein [HDL] and triglyceride [TG]) also did not reveal significant changes when comparing HIV-positive on HAART versus HIV-positive HAART naive groups. However, additional analyses (using established cut-off values for HDL, LDL) showed a significant difference in the proportion of individuals categorized with ‘’low HDL’’ status, i.e. 68% for the HIV-positive HAART naive compared to 40% for the HIV-positive on HAART group (P =0.022). HAART also enhanced anthropometric measures of obesity, with significant differences for weight gain, triceps skin fold (TSF), biceps skin fold (BSF), waist circumference (WC) and mid-upper arm circumference (MUAC) between the naive and HAART groups. This applied similarly for first and second line treatments. Conclusion: The study established the prevalence of several traditional lifestyle CVD risk factors in both HIV-positive naive and HIV-positive on HAART in the Cape Winelands region of South Africa. HAART enhanced several measure of weight gain and lipid profile, suggesting a restoration to health and well-being. However, there was a relatively high prevalence of obesity in the HIV-positive on HAART group (especially females) thus placing them at a greater risk for the onset of future cardio-metabolic complications. We are unable to distinguish whether this risk is due to HAART or lifestyle-related risk factors, and this question requires further investigation. The findings of this study indicate that clinicians should be attentive of lifestyle-related CVD risk factors in HIV-positive persons and make an effort to counsel patients to adopt improved lifestyle choices.
The cellular response of triple-negative breast cancer to short-term starvation: implications for chemosensitivity
(Stellenbosch : Stellenbosch University, 2021-03) Prangley, Charne; Engelbrecht, Anna-Mart; Davis, Tanja; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Introduction: Breast cancer is currently the most common cancer among women globally. Triplenegative breast cancer (TNBC) is an aggressive and often drug-resistant sub-type of breast cancer that is correlated with poor patient outcomes. As a result, adjuvant therapies that may improve drug sensitivity are currently being sought. Due to the unique metabolic hallmarks of cancer, metabolic adjuvant therapies have become an area of increasing interest. We therefore set out to investigate the effect of short-term starvation (STS) on the growth, viability, and metabolism of TNBC cells and a benign breast epithelial cell line. We also investigated the effect of STS on chemotherapyinduced cytotoxicity in these cells to determine whether STS may enhance the effect of doxorubicin in TNBC. Methods: Three cell lines were utilised for this study: a benign breast epithelial cell line (MCF- 12A), and two triple-negative breast cancer cell lines (BT-549 and MDA-MB-231). Western blotting was employed to determine the effect of starvation over time on growth and proliferation signalling pathways (PI3K/Akt) and markers of autophagy (Atg5, p62 and LC3-II). Immunocytochemistry was utilised to quantify autophagic puncta. Cell cycle progression and viability were assessed using flow cytometry and a WST1 assay, respectively. The effect of STS on chemosensitivity was then established by incubating cells in standard or starvation-mimicking media for 24 hours, whereafter they received doxorubicin at a concentration of 2.5 μM. Chemosensitivity was then established in terms of live cell number, cell death and viability, and cell cycle progression. Results and Discussion: In response to STS, the MCF-12A cells downregulated pro-growth signalling pathways, while the MDA-MB-231 cells showed significant upregulation. A 24-hour starvation period had no significant effects on these pathways or on autophagic flux in the BT-549 cells. Both the MCF-12A and MDA-MB-231 cell lines significantly upregulated autophagic flux in response to STS, with the latter achieving the most significant effect at 24-hours. This may have offered protection to these cells, as a period of starvation prior to drug administration reduced doxorubicin-induced G2/M arrest. Additionally, STS had no other significant effects on chemosensitivity in these cells. In the BT-549 cells, however, starvation was able to significantly increase the percentage of dead cells in the group that received STS prior to doxorubicin treatment. As autophagy was not significantly increased in this cell line during starvation, this suggests that autophagy may indeed play a role in drug resistance. Conclusion: In summary, the cell lines which displayed an upregulation of autophagy at 24 hours of starvation were not sensitised to doxorubicin in terms of cell death, and also experienced amelioration of doxorubicin-induced G2/M arrest. This supports the notion that autophagic upregulation may protect cancer cells from doxorubicin-induced cytotoxicity and contribute to drug resistance. However, to gain a more thorough understanding of this phenomenon, future studies investigating the mechanisms by which autophagy promotes chemoprotection are recommended.
Changes in acetylcholine receptor expression : neuromuscular junction morphology and associated myonuclei in BALB/C mice following muscle contusion injury
(Stellenbosch : Stellenbosch University, 2015-03) Louw, Elizabeth Adrienne; Myburgh, Kathryn H.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Contusion injuries cause significant muscle damage, that effects skeletal muscle in its entirety, including the innervating motorneuron and the myofibre’s neuromuscular junction (NMJ). Upon injury, the acetylcholine receptors (AChR) scatter and disintegrate yet reaggregate over time to re-create an optimally functioning motor end-plate. This process involves the upregulation of the receptor subunits’ – α, β, γ, δ, ε – expression to varying degrees. Satellite cells are key role players in muscle regeneration, but studies linking the regenerative roles of satellite cells to the rehabilitating NMJ are limited. Moreover, the majority of studies on acetylcholine receptors investigate the effects of a denervation event rather than an injury that affects the muscle tissues in their entirety. Bromodeoxyuridine (BrdU) is a useful tool in labelling and tracking proliferated satellite cells. Two experimental groups (referred to as PCR and BrdU group) of male BALB/C mice were subjected to a hind limb contusion injury induced with the mass-drop technique. Alzet® mini-osmotic pumps delivering BrdU (50 mM, 1.0 μl.h−1 release rate) were inserted into the BrdU group prior to injury. Animals were sacrificed at days 1, 3, 5, 7, 10 and 14 post injury. Both injured and contralateral, non-injured gastrocnemius muscles were collected. qPCR was performed for AChR-γ and AChR-ε mRNA expression on the muscles of the PCR mice. Muscles from the BrdU group were cryo-sectioned in longitudinal orientation and stained with 1) H&E and 2) immunohistochemically with α-bungarotoxin to visualise AChRs; and also with antibodies against laminin and BrdU. Images were obtained by light microscopy (1) to detect and describe contusion injury in longitudinal section and confocal microscopy (2) to observe the form, prevalence and arrangement of NMJs along both the injured and non-injured muscle. AChRs position themselves into junctional folds that adopted a coral-like appearance – identifiable as a NMJ. A 3D z-stack image at 40x magnification revealed myonuclei residing beneath the NMJ in intimate connection. These NMJ were arranged along the muscle in central band; however contusion injury resulted in a disintegration of part of or the entire junctional complex. Super-resolution microscopy revealed in depth structural arrangement in the intact NMJ. This became jagged and dispersed following contusion injury, by 7 days. Robust, regenerating NMJs were detected in muscle sections at 14 days post injury. Surface area and volume were measured and revealed a trend towards a decrease in NMJ size at 7 days post injury, followed by an exaggerated increase in NMJ size by day 14 post injury. A two-step staining procedure exposed BrdU+ cells residing beneath the neuromuscular junction at 14 days post injury. The results of this study show that NMJ morphology is indeed affected by muscle contusion injury, and repairs itself by increasing its AChR subunit production. We explored novel techniques for analysis of neuromuscular morphology and its changes after injury and during regeneration. We have also ascertained the migration of satellite cells to beneath the NMJ following contusion injury. These findings lay the foundation for future research to better understand the role players involved in neuromuscular regeneration.
Circadian rhythms as novel chemotherapeutic strategies for breast cancer
(Stellenbosch : Stellenbosch University, 2014-12) Mitchell, Megan Irvette; Engelbrecht, Anna-Mart; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Introduction: Mammalian circadian rhythms form an integral physiological system allowing for the synchronisation of all metabolic processes to daily light/dark cycles, thereby optimising their efficacy. Circadian disruptions have been implicated in the onset and progression of different types of cancers, including those arising in the breast. Several links between the circadian protein Per2 and DNA damage responses exist. Aberrant Per2 expression results in potent downstream effects to both cell cycle and apoptotic targets, suggestive of a tumour suppressive role for Per2. Due to the severe dose limiting side effects associated with current chemotherapeutic strategies, including the use of doxorubicin, a need for more effective adjuvant therapies to increase cancer cell susceptibility has arisen. We therefore hypothesize, that the manipulation of the circadian Per2 protein in conjunction with doxorubicin may provide a more effective chemotherapeutic strategy for the treatment of breast cancer. The aims of this project were thus to: (i) Characterize the role of Per2 in normal breast epithelial cells as well as in ER+ and ER- breast cancer cells; (ii) to determine the role of Per2 in doxorubicin-induced cell death, (iii) to determine the role of Per2 in autophagy and finally (iv) to assess whether the pharmacological inhibition of Per2 with metformin, can sensitize chemo-resistant MDA-MB-231 breast cancer cells to doxorubicin-induced cell death. Methods: An in vitro model of breast cancer was employed using the normal MCF-12A breast epithelial, estrogen receptor positive (ER+) MCF-7 and estrogen receptor negative (ER-) MDA-MB-231 breast adenocarcinoma cell lines. Circadian rhythmicity of Per2 protein expression was determined using western blotting, and Per2 cellular localization was assessed using fluorescent confocal microscopy. Per2 was then silenced by means of an endoribonuclease-prepared siRNA, and silencing efficiency was determined with the use of western blotting. The roles of Per2 in doxorubicin-induced cell death and autophagy were assessed by treating MDA-MB-231 breast cancer cells under the following conditions (1) Control, (2) 2.5 μM doxorubicin or 10 nM bafilomycin A1 (3) 30 nM esiPer2 and (4) 30 nM esiPer2 in combination with 2.5 μM doxorubicin or 10 nM bafilomycin A1. Following treatments cell viability was assessed using the MTT assay, western blotting for markers of apoptosis including p-MDM2 (Ser166), p-p53 (Ser15), cleaved caspase-3 and –PARP as well as markers of autophagy (AMPKα, mTOR and LC3). Furthermore, cell cycle analysis, G2/M transition and cell death (Hoechst 33342 and propidium iodide staining) were assessed by means of flow cytometry. The pharmacological inhibition of Per2 was achieved by treating MDA-MB-231 cells with 40 mM metformin as well as in combination with 2.5 μM doxorubicin. MTT cell viability assays, cell cycle analysis (flow cytometry) and western blotting for apoptosis (Per2, p-AMPKα (Thr172), p53, caspase-3 and PARP) were assessed. Results and discussion: A circadian pattern of Per2 protein expression was observed in the normal MCF-12A and MDA-MB-231 cancer cells with protein levels peaking at ±700% and ±500% of baseline was observed. However, no rhythmic expression was observed in the MCF-7 cancer cells. Immunostaining for Per2 showed localization OF Per2 in the cytoplasm as well as in the nucleus of both the MCF-12A and MDA-MB-231 cells. Concentration curves showed a significant reduction in cell viability following 2.5 μM doxorubicin treatment for 24 hours. Per2 protein expression was significantly reduced with both esiPer2 and metformin treatment. Silencing of Per2 in combination with doxorubicin treatment resulted in cell cycle arrest with a significant increase in apoptosis, indicating that Per2 silencing effectively sensitized the MDA-MB-231 cancer cells to the anti-carcinogenic properties of doxorubicin. Modulation of Per2 protein expression was effectively achieved with the use metformin although this decrease occurred independently of AMPKα phosphorylation. A significant increase in apoptosis was observed following treatment with metformin in combination with doxorubicin treatment. However, no changes in cell cycle regulation were observed. Per2 appears to be involved in the regulation of autophagy as a significant increase in autophagy flux was observed when Per2 was silenced. Additionally, this increase in autophagic flux resulted in a significant increase in MDA-MB-231 cancer cell death which was enhanced further when autophagy was inhibited with bafilomycin A1 subsequent to Per2 silencing. Conclusions: Per2 protein expression was shown to display a 24 hour circadian rhythm in the MCF-12A cells, and to a lesser extent in the MDA-MB-231 cells. However, the MCF-7 cells failed to show rhythmic changes in Per2 protein expression. Per2 was shown to be located predominantly in the cytoplasm, with nuclear localization observed when cytoplasmic fluorescent intensity was lower. Per2 silencing effectively sensitized the chemo-resistant MDA-MB-231 breast cancer cells to both doxorubicin-induced cell death and autophagic inhibition.
The comparison between two high-intensity interval training protocols on skeletal muscle and satellite cell dynamics
(Stellenbosch : Stellenbosch University, 2019-03) Sugden, Cameron; Myburgh, Kathryn H.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Introduction: High intensity interval training (HIIT) interventions are popularly used by endurance athletes to increase muscle strength, peak speed and aerobic capacity. Running involves the use of both eccentric and concentric contractions, with the level of the running surface determining the ratio between the two. Downhill running is considered eccentric- biased exercise. Conversely uphill running is considered concentric-biased exercise. Uphill running and downhill running are therefore two different role players in muscle adaptation, although potentially both act through regulating satellite cell (SC) dynamics. Hypothesis: The different modes of HIIT will result in differing skeletal muscle damage, satellite cell activity and morphological adaptation, resulting in differing muscle adaptation, aerobic capacity, muscle strength and running performance. Methods: 12 healthy active males were randomized into either a downhill running (DHG) or an uphill running group (UHG). Subjects underwent baseline and post training performance testing which consisted of a flat VO2max treadmill test, maximal isometric strength test and a 5km road time trial. Training consisted of 10 HIIT sessions over a period of 4 weeks. Each session consisted of 6 intervals at either +5% gradient and 80% peak treadmill speed or -10% gradient and 90% peak treadmill speed. Muscle biopsies and blood draws were taken at baseline, as well as 6 hours after the first and the last session. Results: Performance testing: The UHG, but not the DHG, improved VO2max from baseline (59.48 ± 1.73 ml.kg.min-1 – 61.86 ± 1.28 ml.kg.min-1). The DHG, but not the UHG, improved maximal isometric after the 4 weeks of HIIT (734 ± 133 N - 893 ± 55 N). Both groups improved their 5km TT performance by 3.5 ± 1%. The DHG but not the UHG had a significant increase in CK levels 6 hours after running (p < 0.05). Muscular response to 4 weeks HIIT for the DHG included an increased CSA (p < 0.05), increased SC pool size (0.1 ± 0.001 SC/fibre - 0.3 ± 0.02 SC/fibre), and an increase in myoD after the first bout of exercise (p < 0.05). Muscular adaptations in the UHG included increased capillary to fibre ratio (1.76 ± 0.18 – 2.55 ± 0.20) and capillary density (249 ± 39 mm2 – 304 ± 57 mm2) with training. Conclusion: Four weeks of uphill or downhill HIIT resulted in physiological adaptation by different mechanisms, one by enhanced SC activity and a more forceful contraction and the other involving muscle perfusion and oxygen utilization. The mechanisms of adaptation are training specific, yet they both result in a similar improvement in 5km race performance.
A comparison of compounded-bioidentical hormone formulations versus FDA-approved hormone formulations in breast cancer progression
(Stellenbosch : Stellenbosch University, 2023-03) Mochoele, Kamano Angela; Engelbrecht, Anna-Mart; Africander, Donita; Du Plessis, Manisha; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Introduction: Oestrogen and oestrogen receptor-induced signalling plays an important role in breast cancer development and progression. Studies have shown that certain menopausal hormone therapies {MHTs} containing oestrogens and oestrogens in combination with progestogens, increase the risk of invasive breast cancer. Compounded-bioidentical hormone therapies {cBHTs}, not FDA-approved or regulated by the Medicines Control Council of South Africa, have become a popular MHT and are advertised as safer efficient alternatives. Oestrogen alone and in combination with progestogens such as medroxyprogesterone acetate {MPA} and norethindrone {NET} enhance breast cell proliferation, migration and invasion. lt is therefore important to determine the effects of compounded oestrogen formulations in the development and progression of breast cancer. This study aims to provide a comparative profile of the effects of traditional menopausal therapies {estrone + MPA and estrone + NETA}, an FDA-approved bioidentical formulation {oestradiol + progesterone {bE2+bP4}} with the compounded bioidentical biest hormone formulation E2 + estriol {bE2+bE3} on the progression of breast cancer. Methods: Human ER+ mammary adenocarcinoma cells {MCF7} were used. Proliferation was assessed by determining the cell viability through water-soluble tetrazolium salt {WST-1} assays. The cell cycle was analysed with flow cytometry. Western blot analyses were performed to assess the proliferation marker MCM2, the Pl3K/Akt signalling pathway and epithelial-to-mesenchymal transition {EMT} markers; E-cadherin, N-cadherin, Snail and β-catenin. Migration was measured through a wound healing assay. Results and discussion: All treatment combinations significantly increased cancer cell viability. The cell cycle analysis shows that FDA-approved estrone + MPA and estrone + NETA treatments induced the accumulation of MCF7 cells in the GO/Gl phase of the cell cycle. Western blot analysis revealed that all hormone treatments did not activate the Pl3K/Akt pathway. Furthermore, treatment of BE2 + BP4 indicated mesenchymal characteristics of EMT. The wound closure assay showed that the hormone treatments did not induce migration. Conclusion: According to our findings, there are both similarities and differences among the compounded biest combinations and FDA-approved hormone formulations. Concerningly, cBHT increases cell viability in a manner consistent with the FDA-approved formulations. Similar to FDA- approved therapies, they did not cause migration or activate the Akt pathway for cell proliferation. In contrast, when compared to their FDA-approved counterparts, cBHT formulations exhibited different effects on EMT and the cell cycle. All together these results demonstrate that cBHT treatments did not stimulate the pathways associated with breast cancer progression that was stimulated by the FDA-approved formulations. Future recommendations include investigating the effects of cBHT preparations on other pathways involved in breast cancer initiation and progression in comparison to the FDA-approved formulations.
A comparison of the effect of curcumin treatment on apoptosis, necrosis and autophagy in a MCF-7 mammary adenocarcinoma and a MCF-12A healthy mammary epithelial cell line
(Stellenbosch : University of Stellenbosch, 2009-03) Van den Heever, Martine; Engelbrecht, Anna-Mart; Loos, Benjamin; University of Stellenbosch. Faculty of Science. Dept. of Physiological Sciences.
Breast cancer is currently the primary cause of cancer-related death in women worldwide. Conventional treatments such as radiation and chemotherapy have many deleterious and long lasting side-effects, some of which are permanent, such as infertility. As certain tumour cells can also acquire resistance to chemotherapy, the need for the development of a less severe, yet more effective, targeted anti-cancer treatment exists. Curcumin, a plant polyphenol from Curcuma longa, has long been thought to possess antitumour, antioxidant, anti-arthritic, anti-amyloid, anti-ischemic and anti-inflammatory properties. Numerous studies conducted over the past sixty years confirm this. We aimed at examining the effect of curcumin on cell viability and the different modes of cell death, namely apoptosis, necrosis and autophagy, in the MCF-12A (non-tumorigenic mammary epithelial) and MCF-7 (mammary adenocarcinoma) cell lines. Cells were incubated with different doses of curcumin to evaluate the dose response through a MTT assay. Thereafter, cells were incubated with 200 μM curcumin for 48 hrs and stained with markers and DNA stains for apoptosis (Hoechst, Caspase-3, PARP), necrosis (Propidium Iodide) and autophagy (LC3B and Beclin-1). Cells were examined via fluorescence microscopy, Western Blot- and FACS analyses. MTT results showed no significant decrease in viability in the MCF-12A cell line after curcumin treatment. However, a significant decrease in viability was observed in MCF-7 cells after treatment with 200 μM curcumin (p < 0.05). Treated MCF-7 cells also show clear LC3B expression. FACS results show a significant difference in Hoechst mean fluorescence intensity in MCF-7 cells after curcumin treatment (p < 0.05). This study provides evidence that MCF-7 cells respond to a 200 μM dose of curcumin treatment through metabolic change and induction of the autophagic pathway. The model system used in this study provides groundwork for further cell culture based studies regarding breast cancer and curcumin.

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