Wyndham competition (Oral Presentations)
Identification of novel hypothalamic G protein-coupled receptors involved in the control of reproduction
Genevieve Marie Auger1
Ross Anderson1, Robert Millar1, Claire Newton1
for Neuroendocrinology, Department of Immunology, Faculty of Health Sciences, University of Pretoria1
Introduction: Gonadotropin-releasing hormone (GnRH) secreted from the hypothalamus is the master hormone regulator of the hypothalamus-pituitary-gonadal (HPG) axis, which controls puberty, fertility and reproduction. Several genes have been shown to impact GnRH activity, with defects in these causing reproductive dysfunction including hypogonadotropic hypogonadism (HH). GPCRs are an extremely important family of signalling molecules involved in the majority of endocrine hormone signalling. As a number of GPCRs are expressed in the hypothalamus, there is potential for them to impact the secretion and function of GnRH.
The aim of this project is to characterize whether hypothalamic mutant GPCRs, identified in human patients suffering from HH, cause defective receptor function therefore may indicate a role in the HPG axis.
Methods: Blood from a cohort of European idiopathic HH patients was collected and exome sequenced. Mutations in GPCR genes were identified and shortlisted to those expressed in the hypothalamus. Bioinformatics tools were used to predict whether the mutations would be detrimental to receptor functionality in order to select a subset for further examination. These selected mutant GPCRs were cloned into a mammalian N-terminal epitope-tagging expression vector and their functionality tested in vitro. A receptor ELISA assay was utilized to compare WT/mutant receptor cell surface expression. An inositol phosphate accumulation assay and CRE-luciferase reporter gene assay were used to compare WT/mutant receptor cell signalling
Results: Two GPCRs of interest were shortlisted: Oxytocin receptor (OXR), Neuropeptide Y4 receptor (NPY4R). Bioinformatics analyses showed a 50% chance of the mutant OXR being non-functional and NPY4R 67%.
In vitro studies indicated no significant difference in cell surface expression of the OXR mutants compared to WT.
However, signalling assays suggested a shift in signalling potency, confirming disrupted function.
Conclusions: Further in vitro studies will now be conducted to compare WT/mutant on both mutant OXRs and NPY4Rs in order to confirm their non-functionality. The role of receptors in reproductive physiology will then be examined in vivo. Characterization of novel GPCR mutations implicated in HH will provide insight into the physiological and pathophysiological roles of these receptors in the neuroendocrine control of reproduction.
Understanding the effects of a co-morbid state in the development of early doxorubicin cardiotoxicity
Zaakiyah Emjedi1, Ashwin Isaacs1, Prof Anna-Mart Engelbrecht1
Department of Physiological Science, Stellenbosch University1
Introduction: The clinical use of Doxorubicin (DOX), an effective but potent chemotherapeutic antibiotic, is limited due to its cumulative dose-dependent cardiotoxicity that can lead to the development of congestive heart failure (CHF). One of the leading risk factors of many cardiovascular diseases is overweight/obesity, associated with the consumption of a high fat diet. Interestingly, the overweight/obesity state has been associated with the development of different types of cancers including breast cancer, although the mechanisms still remain unclear.
Considering that overweight/obesity, cancer and CHF are associated with inflammation and oxidative stress, this study aimed to investigate the effects of a co-morbid state (overweight and cancer) in the development of early DOX-induced cardiotoxicity by these mechanisms.
Methods: 4 week old female C57BL6 mice were placed either on a high fat diet (HFD) or standard diet (SD) for 8 weeks. Groups were further divided into two groups were some animals were inoculated with E0771 breast cancer cells (HFD-T or SD-T) and the others were not (HFD-NT or SD-NT). Once tumours were ± 200 mm2, treatment with DOX commenced. Each animal received a cumulative dose of 12 mg/kg DOX every fourth day at a single dose of 4 mg/kg, or Hank’s salt solution which served as a vehicle control.
All injections were conducted intraperitoneally and the animals remained on respective diets for the duration of the study. Four days after the last injection, the animals were weighted, euthanized and their hearts excised. Some tissue was preserved in formalin for histological staining; whereas others were snap frozen for biochemical analyses.
Results: The animals exposed to a HFD were heavier than animals on the SD suggesting that elevated fats contained in diets contributed to weight gain.
However, heart to body weight results demonstrated that tumour groups had a significantly reduced heart to body weight ratio when compared to the groups without tumours. Within these groups, treatment with DOX had no additional effect.
Conclusion: Further experimentation and analysis are required to give a better understanding of the effects of a co-morbid state in the development of cardiotoxicity.
The estrone derivative EE-15-one induces anoikis in breast cancer cells independent of ER status
Tamarin Jurgens1, 2, Michelle Visagie1, Annie Joubert1
Department of Physiology, University of Pretoria1, Centre for Neuroendocrinology, University of Pretoria2
Introduction: Breast cancer still remains the most common cause of cancer related death among women. Cancer cell survival during metastasis depends on the evasion of anoikis or detachment-induced death. 2-methoxyestradiol is an estrogen metabolite which has been shown to have various anti-cancer and antimitotic effects, but its low bioavailability prompted the synthesis of a large number of derivatives.
Aim: Here we investigated the mode of action of one of these, named EE-15-one, as a novel inducer of anoikis in triple negative and estrogen receptor positive breast cancer cell lines.
Methods: The effect of EE-15-one on survival of different breast cancer cell lines was measured by crystal violet staining, while cell morphology was studied using light microscopy. To study the onset of cell death, lactate dehydrogenase (LDH) assays were performed at crucial time points. Adhesion assays were performed to assess the activity of the integrins. Confocal microscopy was used to visualise the actin cytoskeleton and focal adhesions and to study the real-time effects on the actin cytoskeleton and focal adhesions. Signalling protein activity such as extracellular regulated kinase (ERK) and focal adhesion kinase (FAK) was measured using western blot analysis.
Results: EE-15-one induced rapid rounding and detachment of both estrogen receptor positive (MCF-7) and triple negative (BT-20 and MDA-MB-231) breast cancer cells. However, LDH assays revealed that cells start to undergo necrosis only after 24 hours of exposure to EE-15-one. Confocal microscopy showed that focal adhesions were dissolved and the actin cytoskeleton organization was lost within minutes after exposure. Moreover, the activity of the focal adhesion signalling protein FAK was abrogated within 1 hour after exposure while ERK phosphorylation was significantly increased after 1 hour.
Conclusion: Our studies reveal that EE-15-one induces rapid loss of cell attachment and anoikis in breast cancer cells through the dysregulation of the focal adhesions and the actin cytoskeleton independent of the ER status of the cells. Since many cancer cells are resistant to anoikis, EE-15-one may potentially be of great value to induce cell death in otherwise resistant cancer cells.
Characterizing “Sugars” and a preliminary investigation of the effects of its components on memory and behaviour
Yvette Yolanda Chetty1
Panjasaram Naidoo2, Anand Nadar1
School of Laboratory Medicine and Medical Science, University of KwaZulu-Natal1
School of Pharmacy and Pharmacology, University of KwaZulu-Natal2
“Sugars” has been described as an affordable, highly addictive illicit drug cocktail of questionable composition. Data obtained from psychosocial research conducted on addicts has described the ‘roster’ or withdrawal as comprising of symptoms such as runny eyes and nose, hot flushes and cold sweats, severe body pain especially joint pain, abdominal cramps, constipation, goose-bumps, cravings and decreased concentration, which differ from symptoms associated with other illicit drugs. These symptoms are evident every 4 hours. The uncertain composition of the cocktail poses a challenge in providing effective rehabilitation to addicts. It was therefore necessary to identify and quantify the constituents of the cocktail due to the lack of empirical data to substantiate its composition.
Three independently sourced batches of the cocktail were analysed using chromatographic techniques and nuclear magnetic resonance imaging. Although the main compounds identified were heroin, papaverine and noscapine, there was variation noted between sources. The effects of the above compounds on memory and anhedonic behaviour were evaluated using well established behavioural assessments in a C57Bl6 mouse model. In Phase 1, the drugs were burnt on aluminium foil, mimicking the method used by addicts, in a specialised smoke chamber. Animals inhaled these fumes for 5 minutes daily for 12 days, half the group were euthanized and the remaining mice underwent a 10-day withdrawal period (Phase 2). Animals were subjected to Sucrose Preference and Morris Water Maze tests to assess anhedonic behaviour and memory respectively, during both phases of the study. All protocols were approved by the Animal Ethics Committee (UKZN).
For sucrose preference, a one-way ANOVA test showed a significant difference between group means. The papaverine administration group had significantly higher consumption than the Control (withdrawal) and Noscapine (withdrawal) groups. A Mann-Whitney test revealed a significant difference between the Noscapine (administration) and Noscapine (withdrawal) groups for the Morris Water Maze test, however, one-way ANOVA revealed no statistical significance between group means. Data was analysed using GraphPad Prism 5 software (p<0.05). This preliminary study is part of an ongoing study investigating the effects of Sugars on physiological, biochemical and behavioural factors in the mouse.
The impact of different concentrations of levonorgestrel on whole blood viscoelasticity and erythrocyte ultrastructure: a case study
Konrad de Vries
BarendGerhardusLindeque, Albe Carina Swanepoel
Introduction: Use of combined oral contraceptives (COCs) presents with an increased risk of venous thromboembolism (VTE). However, the underlying mechanisms are poorly understood. Nordette® and Triphasil®, two COCs containing ethinylestradiol (EE) at the same concentrations, differ in their concentrations of levonorgestrel (LNG). LNG is a second-generation progestin with known risks for VTE. This case study investigated the effect that COCs containing EE in combination with different concentrations of LNG had on whole blood viscoelasticity, whole blood counts, and erythrocyte sedimentation rate (ESR) as well as biophysical characteristics of erythrocytes.
Methods: Blood was drawn from two healthy females; one using Nordette® and the other using Triphasil®. Both participants were between the ages of 18-35, non-smokers, not using any chronic medication, had no history of thrombotic disease or any known genetic abnormalities and had not used aspirin 48 hours prior to testing. Citrated whole blood collected by a qualified phlebotomist was used for viscoelastic and ultrastructural analysis. The following methods were utilised: whole blood counts were determined using the Samsung Labgeo HC10® haematology analyser; thromboelastography (TEG) was performed to determine the viscoelasticity of whole blood samples, scanning electron microscopy (SEM) was performed to determine morphology and membrane ultrastructure of erythrocytes; and erythrocyte sedimentation rate (ESR) was performed to give an indication of inflammation.
Results: SEM analysis revealed both COC users’ samples had compromised erythrocyte structures, as they lost their normal biconcave, rounded shape. Spontaneous fibrin formation was visualised in the COC users’ samples, which is significant as no thrombin was added during sample preparation. The haematology analyser revealed no diagnostic flags for either participant. TEG analysis showed decreased reaction time to clot formation for both females. ESR was notably higher for both COC users compared to standardised values.
Conclusion: The increased risk of developing a VTE when using a COC containing LNG could be explained at least in part due to the effect that it has on whole blood clot reaction time, erythrocyte ultrastructure along with the inflammatory profile and the presence of spontaneous fibrin formation.
Attenuated metabolic function and oxidative stress in the myocardium - with sugar-sweetened beverages intake at the heart of it all
Dr Danzil E Joseph1, Dr Dirk Bester2, Prof Amanda Lochner1
Stellenbosch University1; Cape Town University of Technology2
Although high intake of sugar-sweetened beverages (SSB) is considered a major contributor to non-communicable diseases, the underlying mechanisms remain unclear. As previous animal studies typically used sugar- or fructose-enriched diets at relatively high dosages, this study aimed to establish an improved experimental system to better model SSB-mediated effects on the heart. We hypothesized that SSB consumption exerts early effects on mitochondrial function together with activation of non-oxidative glucose pathways (NOGPs) - with detrimental effects on the heart leading to oxidative stress and decreased heart function. Male Wistar rats (~200 gr) were gavaged daily with 3-5.1 mL of a commercial SSB for 6 months (~125 mL/day in human terms), respectively, vs. two matched controls (water gavaging, caloric equivalent). Phenotypic changes were monitored and the following parameters evaluated after 6 months: a) circulating metabolite levels (triglyceride, cholesterol, HbA1c, uric acid, fasting glucose); b) oral glucose tolerance tests (OGTT); c) working heart perfusions (regional ischemia); d) in vivo heart functional analysis (echocardiography); e) mitochondrial respiratory function; and f) heart tissue analyses (oxidative stress markers, NOGPs). SSB consumption did not alter weight gain or circulating blood metabolites, although it significantly lowered the area-under-the-curve for OGTTs.
Heart functional assessments demonstrated that SSB intake did not lead to significant changes vs. controls (at baseline and with ischemia), while diastolic blood pressure was moderately lower. However, SSB intake significantly attenuated mitochondrial oxidative phosphorylation (fatty acid oxidation) and electron transport chain activity. Moreover, some NOGPs were downregulated in the heart (polyol pathway, PKC, AGE), while the HBP was upregulated vs. controls.
Thus although SSB intake results in limited phenotypic and heart functional changes, early alterations in the heart include diminished mitochondrial respiratory capacity (fatty acid oxidation) together with HBP activation. As such perturbations are linked to the onset of cardio-metabolic complications, these organisms may be at risk in the longer-term despite an apparently healthy phenotype.
Characterisation of potential novel small molecule therapeutics targeting the growth hormone releasing hormone receptor
Robin Du Preez1
Dr Claire L. Newton1, Dr Ross C. Anderson1, Prof Robert P. Millar1
University of Pretoria
Introduction: Growth hormone releasing hormone (GHRH) is important for the regulation of growth hormone (GH) secretion from the anterior pituitary. Disorders can occur when there is an over or under secretion of GH. GHRH also has other important extrapituitary effects (eg. cancer). It elicits its effects through binding to the growth hormone releasing hormone receptor (GHRHR) which is a G protein-coupled receptor. Although GHRH and GHRHR are important therapeutic targets, no non-peptide therapeutics currently exist targeting the GHRHR and it is therefore the aim of this project is to test putative small molecule
GHRHR-binding compounds to determine their activity at the GHRHR and to further refine and test any identified ‘hit’ compounds.
Methods: Using in silico docking, 50 putative GHRHR interactive compounds have been designed using predicted models of the GHRHR extracellular and transmembrane domains. These compounds have been synthesised and their in vitro agonist and antagonist activity tested using HEK 293-T cell lines expressing GHRHR and a CRE-luciferase reporter assay which responds to cAMP, a second messenger produced upon GHRHR activation. GHRH and the antagonist JV-1-36 were used as controls for GHRHR agonism and antagonism, respectively.
Results: Western blotting confirmed the expression of GHRHR in transfected HEK293-T cells. A CRE-luciferase assay screen identified two agonist compounds which showed a significant level of stimulation at the receptor. No stimulation by these compounds was detected in cells not expressing GHRHR, confirming that the response was specific. Dose response curves provided information on the potency/efficacy of these compounds in relation to GHRH. “In catalogue” screening was then undertaken to identify a further 21 compounds that are structurally related to one of these potential agonists found. The CRE-luciferase assay screen also identified several putative antagonists able to reduce GHRH-induced stimulation of GHRHR more effectively than the peptide antagonist JV-1-36.
Conclusion: In this study, we have identified several small-molecule GHRHR interactive compounds, confirming the validity of in silico docking as an approach for GPCR-targeted drug development. The ‘hit’ compounds will now be further refined through “in catalogue” screening, aiding in the process of producing new effective therapeutic compounds targeting GHRHR.
The significance of mitophagy in myocardial ischaemia/ reperfusion (I/R): the effect of melatonin
Dr Rudwan Salie2, Professor Amanda Lochner1
Division of Medical Physiology , Faculty of Medicine and Health Sciences, Stellenbosch University1
Biomedical Research and Innovation Platform (BRIP), Tygerberg, SAMRC2
Introduction: Mitochondria play a central role in providing cardiomyocytes with a continuous supply of ATP for contractile activity. During I/R mitochondrial ATP synthesis is disrupted accompanied by the generation of reactive oxygen species (ROS). Protection can be induced by removal of damaged mitochondria (mitophagy) or ROS. The aim of this study was to assess the effect of (i) I/R and (ii) melatonin treatment on mitochondrial oxidative phosphorylation (ox phosph) and mitophagy and to evaluate (iii) the role of these changes in cardiomyocyte damage.
Methods: Male Wister rat hearts were perfused ex vivo and exposed 35min regional ischaemia/60min reperfusion and infarct size determined using the tetrazolium method. In another series, hearts were exposed to 20min global ischaemia/30min reperfusion. Hearts were treated with melatonin (50M), pre- and post-ischaemia. After perfusion, mitochondria were isolated for (i) determination of ox phosph using an Oxygraph and (ii) evaluation of mitophagy using Western blotting (PINK-1, Parkin, TOM 70, p62SQSTM and SIRT3).
Results: Melatonin treatment significantly reduced infarct size (ptonin-induced cardioprotection was associated with significant changes in mitophagy markers during stabilization and reperfusion. Whether this is a causal relationship, remains to be established.
The effect of a combined oral contraceptives containing ethinylestradiol and drospirenone on the viscoelasticity of whole blood clots and erythrocyte morphology
BarendGerhardus Lindeque1, Albe Carina Swanepoel1
University of Pretoria1
Introduction: Prescribing a specific combined oral contraceptive (COC) to a female user is based on the dosage of estrogen, type of progestin generation and the dosage of these two compounds combined. Drospirenone (DRSP) is a novel steroidal progestin that has antimineralocorticoid and antiandrogenic effects and is an aldosterone antagonist. The synthetic estrogen 17α-ethinylestradiol (EE) is commonly used in oral contraceptives and is considerably more active than natural estrogens. When DRSP and EE are used in combination in COCs, their properties have seemed to be closer to natural progesterone levels and antiandrogenic and antimineralocorticoid effects. These hormones can cause platelet activation and also lead to a proaggregatable state of platelets. Erythrocytes play a vital role in venous thrombotic embolism in several ways by increasing the viscosity of blood and help direct platelet towards the damaged endothelium. The aim of this study was to investigate the impact of an EE and DRSP containing COC namely Yaz® on the viscoelasticity of whole blood clots and the biophysical characteristics of erythrocytes.
Methods: Whole blood was drawn from 30 healthy females using the oral contraceptive pill Yaz®. Thromboelastography (TEG) was used to investigate the impact of the DRSP and EE containing COC on the viscoelasticity of whole blood clots. Erythrocyte sedimentation rate (ESR) was also done. Biophysical characteristics of the whole blood was analysed by scanning electron microscopy (SEM). Heam-analysis was done to determine whole blood counts.
Results: SEM revealed alterations to erythrocyte shape accompanied by activated platelets and the formation of spontaneous fibrin masses, without the addition of thrombin to initiation coagulation. TEG analysis indicated a shorter clot formation time indicating a possible prothrombotic profile which also correlated to the SEM results seen with the spontaneous fibrin formation. ESR values were in the normal ranges. Heam- analysis indicated that only the lymphocyte counts were lower.
Conclusion: The combination of EE and DRSP as oral contraceptive pill results in a prothrombotic state characterized by alterations to erythrocyte shape, platelet activation and spontaneous fibrin mass formation.
The Effect of Sleep Fragmentation on the Perception of Experimentally-Induced Deep Muscle Pain in Women with Primary Dysmenorrhoea and Healthy Controls
Iacovides S1, Baker FC1,2
Brain Function research Group,School of Physiology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg1
Center for Health Sciences, SRI International, Menlo Park, California, USA2
Introduction: Pain and sleep are believed to have a reciprocal relationship; whereby pain disrupts sleep and disturbed sleep is thought to increase pain sensitivity. The latter part of the sleep-pain relationship is not well-understood, but essential given that poor sleep increases the risk of pain spreading from a localised disorder to a more widespread condition. We aimed to investigate the effect of sleep fragmentation on experimentally-induced pain in pain-free women, as well as in women who experience painful menstruation; dysmenorrhoea. Dysmenorrhoea is believed to be a chronic painful disorder that is not limited to menstruation, as women with primary dysmenorrhoea have been shown to be hyperalgesic.
Methods: 10 women suffering from primary dysmenorrhoea and 9 controls underwent an injection of hypertonic saline into the extensor muscles of the forearm. The women rated the intensity of their pain using Visual Analogue Scales (VAS) every 30 seconds until the pain had completely subsided. Pain assessments were conducted following habitual sleep as well as after one and two consecutive nights of sleep fragmentation; which mimics the disrupted sleep patterns of clinical pain patients. Mood, somatosensory sensation (touch), morning vigilance and sleep quality were assessed.
Results: The controls and dysmenorrhoeic women were matched for age, BMI, and menstrual cycle length, however, the dysmenorrhoeic women rated their menstrual pain significantly higher on the VAS (p < 0.001) and experienced a longer menstruation phase (p = 0.03). Sleep fragmentation led to poorer mood scores, and reduced subjective sleep quality and morning vigilance (p < 0.001). Pain assessments were analysed by calculating area under the curve (AUC) to incorporate pain intensity and duration. No significant differences were found across the nights for both groups. However, after the first night of sleep fragmentation, the AUC for the dysmenorrhoeic women was significantly greater than the controls (p = 0.02). Sleep fragmentation had no effect on touch (p > 0.05).
Conclusion: One night of sleep fragmentation increased the perception of a deep-muscle experimental pain in women with dysmenorrhoea, compared to controls. Sleep fragmentation-induced increased sensitivity is specific to a noxious stimulus, as touch sensation was unaffected by disruption of sleep.
The anti-inflammatory effects of Sutherlandiafrutescens in a cell and animal model
Prof Gill Dealtry1
Department of Biochemistry, Nelson Mandela Metropolitan University1
Chronic low-grade inflammation associated with activation of the immune system, is postulated to be involved in the pathogenesis of obesity-related insulin resistance (IR) and type 2 Diabetes (T2D). Furthermore, studies show that T2D can both induce inflammation, and exacerbate the inflammatory response. Two major macrophage sub-populations involved in the regulation of immune responses are identified. These are the classically activated M1 macrophage sub-population that stimulates inflammation and the alternatively activated M2 macrophage sub-population that shows anti-inflammatory activities and enables wound repair after completion of the inflammatory response. Obese T2D individuals show high levels of reactive oxygen species (ROS), nitrogen species, impaired antioxidant defenses and increased inflammatory adipokines. In these individuals, macrophages are recruited into adipose tissue and are classically activated by the adipokines, contributing to increased inflammation and IR.
A South African medicinal plant, Sutherlandiafrutescens (S.frutescens) is known to have anti-diabetic, anti-inflammatory and anti-oxidant properties. We have shown in a genetically defined diabetic (db/db) mouse model that S.frutescens can reduce total body weight, but does not prevent hyperglycaemia in 13 week old diabetic mice. This indicates that, whilst S.frutescens can reduce obesity, it is not an effective anti-diabetic therapy once T2D is established. However, it may be effective during early stages of T2D and could regulate diabetes-associated inflammation.
This study investigated inflammation related immune responses in the THP-1 human monocyte model system. The effects of S.frutescens on induction of macrophage differentiation (using PMA) and inflammatory activation of the macrophages (using bacterial LPS) were investigated. M2 induction with IL-4 and IL-13 was also examined. S.frutescens significantly increased expression of the macrophage maturation marker CD14 in PMA treated THP-1 monocytes. Differentiated THP-1 macrophages stimulated with the pro-inflammatory agent LPS, showed reduced expression of the M1 CD marker, CD86, and concurrently induced expression of the M2 marker, CD206, following S.frutescens treatment. This indicates that S. frutescens promotes macrophage development, but reduces the inflammatory condition. After IL-4 and IL-13 induction of THP-1 macrophages, the residual CD86 positive population was significantly reduced by S.frutesens, and the CD206 population was maintained, supporting our hypothesised anti-inflammatory properties of S. frutescens.
Evaluation of the neurotoxicity of pentachlorophenol and its active metabolites on sh-sy5y neuroblastoma cells
Stander, B.A1; Steenkamp, V2
Department of Physiology, Faculty of Health Sciences, University of Pretoria1
Department of Pharmacology, Faculty of Health Sciences, University of Pretoria2
Introduction/Aim: Pentachlorophenol (PCP) is an organochloride pesticide, which due to its stable nature remains ubiquitous within the environment, and is classified as a persistent organic pollutant. PCP and its active metabolites, tetrachlorobenzoquinone (TCBQ) and tetrachlorohydroquinone (TCHQ), are neurotoxic in humans, however little is known with regard to the mechanisms thereof. This study evaluates the neurotoxicity of PCP and its active metabolites using SH-SY5Y human neuroblastoma cells as a model.
Methods: An SRB colorimetric assay was used to evaluate cell growth inhibition and to establish IC50 concentrations. Flow cytometry was used to study the effects on cell cycle progression and mode of cell death using propidium iodide (PI) and Annexin V-FITC/PI stains respectively. Acetylcholinesterase (AChE) inhibition was determined using Ellman’s esterase assay.
Results: The IC50’s for PCP, TCBQ and TCHQ were 80, 35, and 66 µM respectively. Cell cycle progression was analysed relative to a vehicle control. For PCP, a G1 phase block was observed after 12 h, and significant increases in sub-G1 populations occurred after 24 and 48 h, with greater cell death indicated at dose-dependent concentrations of PCP. TCBQ and TCHQ exhibited G2/M blocks after 12 h, as well as sub-G1 increases after 24 and 48 h. PCP and TCBQ exhibited greater propensities to induce apoptosis, while TCHQ induced more necrosis. Significant (p < 0.05) inhibition of AChE was observed for the metabolites, however no inhibition was evident at cytotoxic concentrations of PCP.
Discussion/conclusion: Of the three compounds tested, TCBQ was the most potent, and PCP the least, however all three appear more toxic than DDT, a well-defined neurotoxic pesticide (IC50 = 98 µM). Significant (p < 0.05) cell death occurred in a time-dependent and dose-dependent manner. G1 and G2/M cell cycle blocks may have contributed to differing pathways of cell death for each compound. Further clarification of mechanistic differences will be investigated regarding caspase activation, as well as reactive oxygen species (ROS) production, mitochondrial membrane potential and glutathione (GSH) changes. Neurotoxic effects compounded by AChE changes warrant great need for environmental remediation of PCP.
In vitro effects of glutamine deprivation on proliferation and oxidative stress
Prof Anna Margaretha Joubert, Dr Michelle Helen Visagie
Introduction:Tumourigenic cells modify metabolic pathways in order to facilitate increased proliferation and cell survival resulting in glucose- and glutamine addiction. Moreover, tumourigenic cells have an elevated glutamine consumption rate when compared to non-tumourigenic cells. The aim of this study was to investigate the effects of glutamine deprivation in tumourigenic breast cell lines (MCF-7, MDA-MB-231, BT-20) and a non-tumourigenic breast cell line, MCF-10A.
Methods:The effects of glutamine deprivation were demonstrated on cell proliferation, oxidative stress, deoxyribonucleic acid (DNA) damage, cell cycle and apoptosis induction using spectrometry (crystal violet staining), flow cytometry (2,7 dichlorofluorescein diacetate), fluorescent microscopy (anti-8 hydroxyguanosine antibody), flow cytometry (propidium iodide) and flow cytometry (annexin V-FITC).
Results:The MCF-7 cell line demonstrated decreased cell growth to 87%, 80%, 69%, 62% after 24h, 48h, 72h and 96h deprivation from glutamine. The MDA-MB-231 cell line showed 93%, 90%, 88% and 78% cell growth after 24h, 48h, 72h and 96h deprivation from glutamine. Glutamine deprivation resulted in a biphasic response in hydrogen peroxide production where 24h glutamine deprivation resulted in an increased to 1.5 fold and decreased thereafter in a time-dependent manner in the MDA-MB-231 cell line. Hydrogen peroxide levels also increased in a time-dependent manner in the MCF-10A cell line to 1.2 fold. Furthermore, oxidative stress was confirmed with DNA damage following glutamine deprivation. Cell cycle progression studies showed an increase in cells occupying the S phase by 25% in the MCF-7 cell line. Furthermore, after 96 h deprivation from glutamine there was an increase in cells occupying the G2M phase by 11%. Apoptosis studies demonstrated that glutamine deprivation for 48h induced apoptosis in the MCF-7 cell line resulting in decreased viability to 78.91%. Glutamine deprivation resulted in decreased viability to 75% after 96h in the MDA-MB-231 cell line.
>Conclusion:This study demonstrates that glutamine deprivation results in decreased cell proliferation, biphasic ROS generation, DNA damage and apoptosis induction with the estrogen receptor-positive MCF-7 cell line most prominently affected. Furthermore, this study contributes to knowledge regarding the effects of glutamine sensitivity in breast cancer cells that may result in improved pre-sensitization strategies in future chemotherapeutic treatments.
Presensitization with a novel estrone analogue prevents tumour neovascularization in an in vitro radiation-treated bone metastasis model
Jolene Michelle Helena1
Mabeta P1, Coetzee M1, Lakier R2, Nolte E1, Verwey M1, Moosa S1, Etsebeth M2, Sebopa L2, Mercier AE1
Department of Physiology, Faculty of Health Sciences, University of Pretoria1
Department of Radiation Oncology, Steve Biko Academic Hospital, Pretoria2
Introduction: Establishment of the metastatic micromilieu relies on neovascularization for tumour survival. Common treatment modalities of secondary tumour sites include chemo- and radiotherapy which are accompanied by various adverse effects. 2-Ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16) is a novel in silico-designed 2-methoxyestradiol (2-ME2) analogue aimed at improving the pharmacokinetic profile of the parent compound. The aim of this study was to investigate the effect of ESE-16 when used as a radiosensitization agent in an in vitro bone metastasis model representing the individual components, namely primary breast (MDA-MB-231)- and prostate (DU 145) tumour-, osteoblastic (MC3T3-E1)- and osteoclastic (RAW 264.7) bone- and endothelial (HUVEC) cells.
Methods: Spectrophotometric assays determined the half maximal growth inhibitory concentration (GI50) of ESE-16 on all the cell lines. Experimental setup comprised of exposing cells to a non-lethal dose of ESE16 for 24 hours prior to 4 Gy radiation. Apoptosis induction and cell cycle distribution was studied using flow cytometry. Endothelial cell migration and invasion assays were performed on an xCELLigence platform. Tartrate-resistant acid phosphatase (TRAP) activity and actin ring formation were investigated in bone cells. Micronuclei analysis quantified deoxyribonucleic acid (DNA) damage via Giemsa staining. Western blot analyses of angiogenic protein-,bone morphogenetic protein 7 (BMP-7)- and matrix metalloproteinase 9 (MMP-9) expression were performed.
Results: Comparative cytotoxicity studies determined GI50 values for ESE-16 atnanomolar concentrations in all cell lines except the osteoblastic MC3T3-E1 cells.Significant decrease in HUVEC cell migration was observed when cancer cellconditionedmedia was used as a chemoattractant. RAW 264.7 macrophages retainedtheir ability to differentiate into osteoclasts after treatment exposure. Micronucleiquantification indicated retarded DNA repair in both pre-sensitized cancer cell types.Western blot analyses elucidated potential signalling pathways involved in metastaticmicromilieu establishment and maintenance.
Conclusion: Low-dose ESE-16 pre-exposure rendered the treated neoplastic cellsmore susceptible to radiation damage, as well as significantly decreasing endothelialcell migration whilst preferentially sparing the bone cells. This phase 0 studyestablishes proof of concept that ESE-16 may potentially serve as a radiosensitizingagent to improve the clinical outcomes of radiation therapy at bone metastasis sites.Future studies aim at testing this hypothesis in integrated metastases models and invivo experiments.
Obstructive sleep apnea: severity in middle-aged males
Janse van Nieuwenhuizen1
Tjallinks, B2; Nel, JD2; Du Toit, PJ1
Department of Physiology, Faculty of Health Sciences, University of Pretoria1
Clinical Neurophysiologist/Technologist, Private practice2
Introduction: Sleep apnea is classified as a sleep disorder characterised by a complete cessation, known as apneas, or impairment (partial cessation) of breathing, known as hypopneas. During Obstructive Sleep Apnea (OSA), the airways become blocked, mostly due to the collapsing of the throat muscles or increased adipose tissue surrounding the airway. Apneas/Hypopneas lead to intermittent hypoxia and thus to fluxuations of oxygen-haemoglobin saturation (SpO2) throughout the night. This indirectly leads to the over-activity of the sympathetic nervous system. These events lead to a very interrupted sleep pattern and architecture. The most common symptoms of OSA are excessive daytime sleepiness, morning headaches and concentration problems. OSA may lead to various other diseases such as Hypertension, Diabetes Mellitus, Depression and Heart Failure when not treated.
Methods: Patients (n=69) suspected of having OSA that were referred to a private practice of clinical neurophysiology by their primary/specialized physicians, were admitted at a sleep laboratory, based in Pretoria, for a single night. Inclusion criteria is male patients between the ages of 45 and 65 (n=40). Full polysomnography (PSG) was used to monitor the patient’s sleep. Various information such as the desaturation index, amount of obstructive sleep apneas/hypopneas, sleep architecture and baseline oxygen-haemoglobin saturation were interpreted from the test. This information is then used to assess the severity of the disorder in the patient. The PSG data were then compared with other aspects of the patient such as BMI, medical history and questionnaires.
Results: Among patients (n=40, 100% male) the mean BMI is 34.61 (SD ± 6.44) and the mean A/H index is 46.28 (SD ± 26.88). A positive correlation was found between the amount of apneas and BMI using the Pearson correlation coefficient and a significant negative correlation between BMI and baseline SpO2 (mean = 88.99, SD ± 2.82) was found (r = -0.43, p < 0.05).
Conclusion: We can conclude that the baseline SpO2 has a moderate negative correlation with an increase in BMI and that there is a positive correlation between the amount of apneas (A/H Index) and BMI.
Using 3D spheroids as a compound testing platform for cancer drug development
Annie Joubert1, Iman van den Bout2
Department of Physiology, University of Pretoria1; Centre for Neuroendocrinology, University of Pretoria2
Triple negative breast cancers (TNBC) form around 15% of all breast cancers. Typically, TNBC patients have a poorer prognosis due to their resistance to anti-estrogen treatment. 3D cell culture methods have enabled researchers to generate an in vitro environment that closely mimics the in vivo environment of small tumours. The aim of this study was to investigate whether TNBC cells, specifically using the tumorigenic, non-metastatic BT-20 cell line, grown in 3D could be used as a reliable platform for 3D drug discovery and testing. The system was tested by analysing cell death and survival and morphology using microscopy after exposure to novel antimitotic estradiol derivatives as well as classic chemotherapeutics.
The ability of compounds to induce cell death was compared in monolayers versus 3D BT-20 spheroids, which form compact spheroids consistently yet are seldom used in 3D cell culture systems. The effects of the compounds were assessed in monolayer by crystal violet staining and in spheroids by measuring spheroid volume over time. Furthermore, we performed confocal microscopy to visualize both alive and dead cells using fluorescein diacetate and propidium iodide as well as phalloidin-TexasRed to assess any actin cytoskeleton changes.
Crystal violet staining showed that all compounds induced some level of cell death within monolayers. For instance, compound EE-15-one caused rapid cytotoxicity in 2D monolayers. However, this compound had no effect at all on spheroid volume while microscopy showed no increased PI staining indicating no cell death. In contrast, compound ESE-15-one induced inhibition in monolayers but was as effective at inhibiting cell growth in spheroids as colchicine, a classic chemotherapeutic.
Using the spheroid system, we have been able to determine that while EE-15-one is cytotoxic to cells in monolayers it is ineffective in killing cells within spheroids. On the other hand, ESE-15-one affects cells in monolayer to some extent but is effective at inducing cell death in spheroids. ESE-15-one, a microtubule destabilising drug causes a breakdown of the actin cytoskeleton. The 3D spheroid system of the TNBC cell line BT-20 is proving to be useful in identifying compounds that may be effective in killing cancer cells in vivo as opposed to monolayers. Moreover, it is providing an amenable platform for determining the mode of action of new compounds within in a more in vivo-like setting.
Unsaturated fatty acids inhibit osteoclastogenesis through GPR120/β-arrestin 2 signalling pathways
Marlena C Kruger2, Magdalena Coetzee3,4
Department of Physiology, Faculty of Health Sciences, University of Pretoria1
School of Food and Nutrition, Massey Institute of Food Science and Technology, Massey University, Palmerston North, New Zealand2
Department of Physiology, Faculty of Health Sciences, University of Pretoria3
Associate of the Institute for Food, Nutrition and Well-being, University of Pretoria4
Bone is a dynamic tissue that is remodelled continuously by bone forming osteoblasts and bone resorbing osteoclasts. When receptor activator of nuclear factor kappa B ligand (RANKL), a soluble factor produced by osteoblasts, binds to the RANK receptor on osteoclasts precursors, they differentiate and fuse into large multinucleated bone resorbing osteoclasts. Binding of RANKL to RANK activates NF-κB and mitogen-activated protein kinase (MAPK) pathways leading to the expression of osteoclast specific genes. We have previously reported the anti-osteoclastogenic effect of polyunsaturated fatty acids (PUFAs) such as the ω-6, arachidonic acid (AA), the ω-3, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and the ω-7 monounsaturated FA (MUFA), palmitoleic acid (PLA). However, the mechanism of action of these fatty acids in osteoclasts is not fully understood. G-protein coupled receptor (GPR) 120 is a fatty acid receptor that is expressed in a host of tissues including bone. The aim of this study was to determine whether the effects of these unsaturated fatty acids in osteoclasts are mediated by GPR120.
RAW264.7 macrophages were transfected with GPR120 or control shRNA and exposed to RANKL with or without FAs. Osteoclastogenesis was then assessed by tartrate resistant acid phosphatase (TRAP) staining and counting TRAP positive cells with 3 or more nuclei. β-arrestin 2 and Gαq are two signalling pathways that can be activated by GPR120 signalling. β-arrestin 2 and Gαq were silenced using siRNA and signalling pathways were evaluated by western blotting and immunoprecipitation after exposure to FAs and RANKL.
All FAs tested in this study inhibited osteoclast formation and signalling pathways in control shRNA cells. GPR120 silencing did not inhibit AAs effect on osteoclasts. The inhibitory effect of DHA, EPA and PLA on osteoclastogenesis was mitigated by GPR120 silencing. Furthermore, silencing of Gαq did not prevent inhibition of RANKL signalling pathways. However, both GPR120 and β-arrestin 2 silencing prevented the inhibitory effect of DHA, EPA and PLA on RANKL signalling pathways.
These results provide evidence that the anti-osteoclastogenic effect of DHA, EPA and PLA may be mediated through GPR120/β-arrestin 2 pathways. It also demonstrates that PLA may behave similar to ω-3 PUFAs in bone.
Investigating the effect of HIV exposure on immunological and neurological development in exposed but uninfected infants through head circumference and interferon-beta measurements
Jacobus Johannes Le Roux
Theresa Rossouw, Peet Du Toit
Department of Physiology, University of Pretoria
Objective: Conduct a pilot study to assess the association among HIV exposure, head circumference (HC) and Interferon-Beta (IFN-β). HC has been shown to correlate with brain size and cognitive function in later life. IFN-β is known to play a significant  role in a host’s acute anti-viral response. Elevated levels of IFN-β have been found in certain neurological abnormalities, suggesting that sustained elevation of IFN-β levels might lead to neurodevelopmental delay and a decrease in HC.
Method: Fifty-five women and their infants were recruited at Kalafong hospital and paired blood samples taken at birth and 10 weeks post-partum. Twenty mother-infant pairs with adequate samples were included in a sub-analysis exploring IFN-β levels in infants, tested by means of manual ELISA. HC was categorised according to percentiles and z-scores. Analysis was performed in STATA 14 and significance set at 5%.
Results: Fifteen mothers were HIV-infected and 5 uninfected. Of the infected mothers, all were on antiretroviral therapy and 60% had an undetectable HIV viral load. Exploratory statistics showed no difference in terms of maternal age, parity, body-mass index, or gestational duration between the groups. HIV-exposed infants were similar in weight and length, but had significantly smaller HC than their unexposed counterparts at birth (p=0.026) but no longer at 10 weeks (p=0.26). HC was associated with infant weight at birth (p=0.02) but not with APGAR scores at 1 and 5 minutes. No associations were found between HIV-exposure status and IFN-β or between HC and IFN-β at either time point.
Conclusion: HIV-exposed infants had a significantly smaller HC at birth than unexposed infants. No associations were found between IFN-β levels in infants and exposure status or HC.
Explanation: These results confirm previous studies that have shown lower HC in HIV-exposed infants. This could, however, not be explained by the hypothesis that increased levels of IFN-β are responsible for this observation. A low HIV viral load and hence limited viral antigen exposure of the infant, the influence of other concomitant viral infections that were not tested for e.g. cytomegalovirus, or the lack of test sensitivity could be some factors that affected the results.
Muscle-damaging exercise, exosomes and microRNA
Dr Peter Durcan1, Professor Kathryn H Myburgh1
Background: Exosomes (Exo) are nano-sized mediators of intercellular communication. Exo contain diverse biomolecules and are stable within biofluids. Circulating Exo, suspended in blood, transfer encapsulated information between distant tissues. Recent evidence suggests that circulating Exo profiles adapt to differing physiological and pathological states., Despite the known active role of skeletal muscle (SkM) as a systemic communicator, there is a paucity of information on Exo and Exo content arising from (SkM), particularly within the context of exercise.
Aims: To determine the size and number of Exo, as well as encapsulation of selected muscle-enriched microRNAs (myomiRs) in Exo, in response to an acute bout of muscle-damaging exercise.
Methods: The exercise intervention constituted a combination of plyometric jumping and downhill running. Serum creatine kinase activities (CK) and Exo were analysed from blood samples of 9 subjects at rest and 2 and 24 hr post-exercise. Perceived muscle pain (PMP) was assessed on a scale of 1 to 10 at 2, 24 and 48 hr post-exercise. Plasma Exo were isolated using size exclusion columns and visualised using transmission electron microscopy (TEM). Exo size and number were quantified using nanoparticle tracking analysis (NTA), and miR expression was quantified with qPCR, with normalisation to an exogenous control (cel-miR-39).
Results: PMP and CK were significantly elevated post-exercise, providing indirect evidence for muscle damage. A simplified protocol for fast exosome visualisation using TEM was achieved, and revealed an abundant and heterogeneously-sized pool of intact Exo. A concomitant abundance of Exo was seen with NTA (mean = 9 x 1010 particles/ml plasma). Mean Exo diameters were 127 ± 15 nm across all timepoints. No change in Exo size or number was seen over time. Detection of the SkM-specific miR-206 was found to be variable both at baseline and post-exercise. No myomiRs in Exo changed across timepoints. However, Exo miR-31 decreased at 24 hr post-exercise when compared to baseline (p < 0.05).
Conclusion: Rather than a change in Exo size or number, the miR-31 cargo of Exo decreases post muscle-damaging exercise. These data substantiate the premise that circulating Exo carry selectively packaged cargo that changes during differing physiological states.
Increases in Carotid Intima-Media Thickness Are Not as Extensive in Younger as Compared to Older Individuals with Stroke in Africa
Eitzaz Sadiq1, Angela J Woodiwiss1, Angela J Woodiwiss1
School of Physiology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg1
Background: Ischemic stroke occurs in those 15-to-20 years younger in Africa than in other regions (average age=50 as opposed to 65-70 years). Whether this reflects the impact of a more rapid development of atherosclerosis following poorly controlled risk factors or a greater chance of arterial occlusion in the presence of a lower degree of atherosclerosis, is uncertain.
Methods: We compared the extent to which carotid IMT (B-mode ultrasound) is increased in 66 patients trols (0.585±0.0.105 mm), patients with stroke 22 p01). Similarly, as compared to age-matched controls (0.0.722±0.130 mm), patients with stroke ≥50 years of age had an increased IMT (0.835±0.151, pAlthough an increased carotid IMT associates equally as strongly with stroke in younger as compared to older age groups in Africa, the extent of the IMT change is markedly lower in younger as compared to older stroke patients. These data suggest that either age-related increases in IMT have little pathophysiological significance in Africa, or that younger patients with stroke In Africa may have a greater chance of arterial occlusion in the presence of a lower degree of atherosclerosis.
Differential Carotid Atherosclerotic Changes in Human Immunodeficiency Virus Positive Versus Negative Patients with Critical Limb Ischaemia and Stroke
EitzazSadiq, TaalibMonareng, Andrea Kolkenbeck-Ruh, Tshegofatso Motau, Pitchou Z Gazwa, TalibAbdool-Carrim, Olebogeng HI Majane, Gavin R Norton, Girish Modi, Angela J Woodiwiss
Schools of Physiology (NM, AK-R, TM, PZG, OHIM, GRN, AJW) and Medicine (ES, TM, TA-C, GM), Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa and Department of Physiology, SefakoMakgatho Health Sciences University, Pretoria, South Africa (OHIM).
Introduction: Human immunodeficiency virus (HIV) infection is a recognized risk factor for occlusive arterial diseases, including peripheral arterial diseases and stroke. Whether atherosclerotic changes contribute to a similar extent in HIV versus non-HIV-associated occlusive arterial diseases is unknown. We therefore compared the extent to which carotid atherosclerosis is increased in HIV positive and negative patients with critical limb ischaemia (CLI) and ischaemic stroke.
Methods: 209 Patients with stroke, (46 HIV +ve), and 166 patients with CLI (33 HIV +ve) were recruited from the Charlotte-Maxeke-Johannesburg Academic Hospital. 233 age-. sex- and ethnicity-matched healthy participants (controls) were recruited from SOWETO. The extent of carotid atherosclerosis was assessed from intima-media thickness (IMT) and plaque measurements (B-mode ultrasound).
Results: As compared to age and sex-matched controls, carotid IMT (0.79±0.01 vs 0.69±0.01 mm, p=0.001), but not carotid plaque (23,1 vs 18.4%) was increased in HIV-ve patients with stroke, In contrast, as compared to age and sex-matched controls, carotid plaque (30.4 vs 11.3%, p<0.005), but not carotid IMT (0.65±0.01 vs 0.64±0.02 mm) was increased in HIV+ve patients with stroke. Although as compared to controls, carotid IMT (0.82±0.01 vs 0.77±0.01 mm, p<0.05) and plaque (46 vs 32%, p<0.05) were increased in HIV-ve patients with CLI, HIV+ve patients with CLI failed to show increases in either carotid IMT (0.70±0.01 vs 0.69±0.01 mm) or plaque (27 vs 19%).
Conclusions: In HIV, the extent of atherosclerosis associated with stroke and CLI is markedly altered as compared to non-HIV-related stroke and CLI. Therefore, in HIV, atherosclerotic vascular changes responsible for occlusive arterial events may differ markedly from those in non-HIV-associated events.
Modulating Effects of Green Rooibos (Aspalathuslinearis) Extract on Vascular Function in Obese Wistar Rats
Huisamen B1,2 and Windvogel SL1
Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University1
Biotechnology, Research and Innovation Platform, SA-MRC, Tygerberg2
Introduction: Obesity is a major risk factor for the development of cardiovascular disease, a leading cause of death worldwide. It is characterised by endothelial dysfunction (ED) via a reduction in nitric oxide bioavailability, and an increase in oxidative stress and inflammation, disrupting vascular homeostasis. Rooibos (Aspalathuslinearis), is rich in polyphenols such as aspalathin, and its health promoting properties include antidiabetic, anti-inflammatory, antioxidant, anti-obesity and cardiovascular benefits. The aim of this study was to investigate the ameliorative effect of Afriplex GRTTM, an aspalathin-richedAspalathuslinearis extract, on vascular function in diet-induced obese Wistar rats.
Methods: Adult male Wistar rats were randomly divided into 5 experimental groups (n=10/group) and fed a control or high fat diet (HFD), to induce obesity for 17 weeks. Rats in the HFD and control groups received the aspalathin-riched extract supplementation at 60 mg/kg/body weight from week 11. A Captopril (50 mg/kg/ body weight) group was included as a positive control. Body weight was measured thrice weekly. Oral glucose tolerance tests were conducted during week 16 and intraperitoneal fat content was measured upon sacrifice. Vascular function was determined by subjecting the isolated thoracic aortas to phenylephrine-induced contraction (100 nM¬-1 µM) followed by acetylcholine-induced relaxation (30 nM-10 µM). Blood pressure measurements were determined non-invasively using the CODATM tail-cuff.
Results: HFD rats had significantly increased (p<0.05) body weight and intraperitoneal fat weight compared to controls. Glucose homeostasis was impaired in HFD rats and this was significantly (p<0.05) abrogated by the aspalathin-riched extract supplementation. At baseline, the controls had an enhanced contractile response (p<0.01) compared to HFD group. The aspalathin-riched extract supplementation significantly increased vasodilation (p<0.001), and decreased vasocontractility (p<0.001), systolic and diastolic blood pressure in the HFD group compared to the controls (p<0.001 and p<0.01, respectively).
Conclusion: Afriplex GRTTM may be a potential therapeutic agent against obesity-related vascular dysfunction, impaired glucose homeostasis and hypertension.
The effect of ischemia on the protein phosphatases:Comparing heart and cancer cells
Dr Derick Van Vuuren
Introduction: Ischaemia is characterised by the lack of oxygen delivery to the cells; causing cellular injury and death. Some cell types are better adapted to this type of stress than others. As a response mechanism, cells can depend on reversible protein phosphorylation as a key feature to adapt to their changing environment. In this study, we will aim to investigate the role of protein dephosphorylation, mediated by protein phosphatases, in the response to ischaemia by comparing cells considered to be sensitive to ischaemia (heart cells) with cells resistant to ischaemia (cancer cells).
Methods: To evaluate whether the metabolic stress causes any change in phosphatase activity and expression; ischaemia was simulated in H9c2 cells (cardiomyoblasts) using a modified Esumi buffer in conjugation with a 2-hour incubation in 0% oxygen. Phosphatase activity was measured using pNPP assay, and different common phosphatase inhibitors utilized to further analyse the contribution of individual phosphatases (20mM sodium fluoride was used to inhibit the serine/threonine phosphatases (PSPs), 5 mM EGTA inhibited PP2B and 10 mM sodium orthovanadate inhibited the tyrosine phosphatases). In addition, the expression of protein phosphatase 1, 2A and 2B was measured using standard Western Blotting. Experiments were repeated more than three times, in triplicate. A similar approach will be employed in MDA-MB 243 cells (a breast cancer cell line).
Results: The inhibitors used significantly reduced the measured phosphatase activity 0001in comparison to control. Under normoxic conditions we found that the PSPs contributed 30% of the phosphatase signal in H9c2 cells, with PP2B contributing > 50% of that. There was no statistically significant difference in phosphatase activity between normoxia and simulated ischaemia (SI) (Control: 0.721±SEM vs SI: 0.7459±SEM, unpaired T-test, p>0.05). Moreover, there was no difference in protein expression between control and SI.
Conclusion: Interestingly, phosphatase activity and expression remained the same when exposed to ischemia. The PSPs contributed less to phosphatase activity in the H9c2s than reported in differentiated heart tissue. Moreover, literature reported PP1 as the most abundant and active PSPs. However, our results suggest that PP2B activity contributed more than 50% of signal in PSPs activity. Investigations on the cancer cells are ongoing.
Hepato-protective effects of Amaranthushybridus leaf extract on cadmium-induced liver damage in rats
Constance R Sewani-Rusike1
Walter Sisulu University, Faculty of Health Science, Department of Human Biology (Physiology)1
Introduction: Cadmium (Cd) induces the generation of reactive oxygen species resulting in damage to various organs. Amaranthushybridus (Ah), a leafy vegetable consumed in the Eastern Cape, is reported to possess antioxidant properties. Although in vitro antioxidant potential of Ah has been widely reported, there are no reports on its protective role on Cd-induced liver damage. This therefore aimed to investigate the protective effects of Ah on Cd-induced hepatic damage.
Methods: Male Wistar rats treated over 4 weeks were divided into 4 groups (n=5/group): Untreated Control; CdCl2 only (5mg/kg orally administered in 1ml); CdCl2 simultaneously treated with Ah (200mg/kg orally administered in 1ml); and Ah. Parameters monitored were: Animal well-being; Glucose tolerance and liver glycogen; total plasma cholesterol; total plasma protein; plasma total antioxidant capacity (TAC) by the FRAP method; liver homogenate TAC by the FRAP and TEAC methods; MDA levels for lipid peroxidation; superoxide dismutase activity and liver histology.
Results: Results showed that treatment with Ah alone resulted in improved glucose tolerance, lowered total plasma cholesterol and lower lipid peroxidation (P<0.05) as well as an increase plasma TAC (P<0.001) compared to untreated controls and Cd treated group, with no effect on animal well-being, homogenate TAC and SOD activity. Treatment with CdCl2 alone resulted in compromised glucose tolerance, reduced liver glycogen, increased plasma protein, SOD activity and lipid peroxidation (P<0.05) compared to untreated controls. Treatment with Ah in the CdCl2 exposed group showed reversal of these parameters compared to control levels. Histologically, all groups showed similar liver architecture. However, at high power, large nuclear dysplasia was observed in CdCl2 treated rats, consistent with genotoxic effects. This effect was partially reduced by Ah treatment.
Conclusion: A. hybridus possesses hypolipidemic properties, as well as protective effects to Cd-induced oxidative and genotoxic liver damage in male rats.
Metabolic reprograming and cancer resistance: do cancer-associated fibroblasts contribute?
Prof. Anna-Mart Engelbrecht1
Department of Physiological Sciences, Stellenbosch University1
Myofibroblasts, often described as “activated fibroblasts” are found to be one of the most abundant mesenchymal cell types present in human carcinomas. It has been postulated that these cells may to promote angiogenesis and stimulate epithelial cancer cell growth. Recent evidence suggests that nutrient deprived epithelial cancer cells present within solid tumours are able to survive these conditions, as a result of their ability to undergo extensive metabolic reprogramming and exploit the metabolic capacities of surrounding cancer-associated fibroblasts (CAFs). Based on this evidence, we hypothesis that the manipulation of CAF metabolism may sensitize nutrient deprived breast cancer cells to doxorubicin-induced cell death. E0771 conditioned media was generated following glucose deprivation and used to treat mouse embryonic fibroblasts (MEF).
The activation of a CAF phenotype was assessed by means of Western blot and confocal microscopy for markers of epithelial-to-mesenchymal transition. Furthermore, cell viability, oxidative stress, glucose uptake, GLUT4 translocation, L-lactate, and autophagy were assessed. MEF conditioned media was then used to treat glucose deprived E0771 cells. L-lactate, ATP, and cell death by means of flow cytometry were assessed.
The effects of CAFs on chemotherapy resistance and metastasis was assessed by treating E0771 cells with 2.5 μΜ doxorubicin in combination with MEF conditioned media and cell viability, apoptosis and migration assessed. Treatment of MEFs with E0771 conditioned media resulted in a significant increase in the mesenchymal markers vimentin and α-SMA. 2-NBDG glucose uptake was significantly increased in conjunction with an increase in the fluorescent intensity of the HA-GLUT4-GFP construct following treatment.
Furthermore, treatment of glucose deprived E0771s with MEF-CM in combination with doxorubicin resulted in a significant increase in mitochondrial reductive capacity in comparison to doxorubicin treatment alone accompanied by enhanced migratory capacity. Our data suggests that glucose deprivation induces a state of oxidative stress in the E0771 cells which is transferred MEFs leading to the “activation” of a cancer-associated fibroblast phenotype. Resulting in increased GLUT4 mediated glucose uptake and increased autophagy culminating in enhanced cancer cell survival and resistance to doxorubicin-induced cell death.
The impact of different classification criteria on the prevalence of diastolic dysfunction and its associated risk factors in patients with rheumatoid arthritis
Sule Gunter2, Chanel Robinson2, Patrick Dessein3
Cardiovascular Pathophysiology and Genomics Research Unit, School of Physiology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg1
Genomics Research Unit, School of Physiology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg2
Rheumatology Division, VrijeUniversiteit Brussel (VUB), UniversitairZiekenhuis Brussel (UZB), Belgium3
Background: Heart failure contributes to the excess mortality experienced by patients with rheumatoid arthritis (RA). Left ventricular (LV) diastolic dysfunction (DD) is the most common cause of heart failure in patients with a preserved ejection fraction, especially frequent in RA. However the diagnosis of diastolic dysfunction in RA is inconsistent and whether the different diagnostic criteria alter the prevalence of DD and its associated risk factors is unclear.
Objectives: This study aimed to identify potential determinants of LV diastolic dysfunction based on different criteria in patients with RA.
Methods: LV diastolic function was measured by echocardiography and determined by the ratio of early-to-late transmitral blood flow velocity (E/A), the ratio of E to the mean of the lateral and septal wall myocardial tissue lengthening at the mitral annulus (e’)(E/e’), and the lateral e’ in 176 patients with RA without overt heart failure. Relationships of comprehensively evaluated traditional cardiovascular risk factors and RA characteristics with markers of LV diastolic function and diastolic dysfunction (as determined by previous and current criteria) were determined in confounder adjusted multivariate and logistic regression models respectively.
Results: Disease duration was associated with all markers of diastolic function (E/A, lateral e’ left atrial volume index all p<0.005), except for E/e’. Age (partial r = 0.19, p=0.01) and diastolic blood pressure (DBP) (partial r=-0.16, p=0.04), but not any RA characteristics were related to log E/e’. The prevalence of diastolic dysfunction was markedly different when applying the old (59%) compared to the new (22%) criteria. When entering all significant variables into the logistic backward regression model, WHR was independently associated with DD when applying the new criteria (OR=3.30, 95%CI=1.70-6.38, p=0.0004). However when using previous criteria, DBP was inversely associated with DD (OR=0.68, 95%CI=0.47-0.99, p=0.05).
Conclusion: Modifiable traditional cardiovascular disease risk factors and disease characteristics are consistently associated with left ventricular diastolic function in RA. However the diagnostic criteria used to identify diastolic dysfunction materially alters the prevalence and associated the risk factors.
Rooibos (Aspalathuslinearis) tea extracts decreases osteoclastogenesis and bone resorption in RAW 264.7 murine macrophages, in vitro
Abe Kasonga, Dr Vishwa Deepak, Sumari Marais
University of Pretoria
Introduction: Bone remodeling is a physiological process that involves thecoordinated synthesis of the bone matrix by osteoblasts and resorption of bone by osteoclasts, thereby creating a regulating cycle. Uncoupling of this process may ultimately cause a variety of bone disorders such as osteoporosis.Rooibos (Aspalathuslinearis) is a caffeine-free tea, rich in polyphenols and antioxidants which have shown beneficial effects in health. In this study, the in vitro effects of aqueous extracts of fermented and unfermented rooibos tea were examined on osteoclast formation and activity in RAW264.7 murine macrophages.
Methods: RAW264.7 murine macrophages were seeded at 15 000 cells/cm2 in the presence of sterile distilled water (vehicle control) or tea extracts (62.5 – 500 µg/ml). Cell viability was determined by alamar blue assay after 48hr exposure. Osteoclastogenesis was stimulated by the addition of RANKL and evaluated after 5-7 days by staining for tartrate resistant acid phosphatase (TRAP) and quantification of TRAP-positive multinucleated cells. Actin ring formation was determined by fluorescent stain using a phalloidin conjugate. Bone resorption assays were conducted on osteoassay plates coated with inorganic synthetic bone surface and after the culture, the cells were washed off to visualize resorption pits. The expression of genes involved in osteoclast formation and activity were assessed by real time PCR. NF-κB activation was determined by secreted alkaline phosphatase (SEAP) assay after stably transfecting cells with a NF-κB inducible SEAP reporter plasmid.This activity was further elucidated using western blotting. Three independent experiments were conducted in triplicate for each test.
Results: Both fermented and unfermented rooibos tea extracts dose-dependently inhibited osteoclast formation and TRAP activity which was accompanied by reduced bone resorption and disruption of characteristic cytoskeletal elements of mature osteoclasts, without cytotoxicity. Rooibos tea extracts decreased expression of key osteoclast specific genes, matrix metalloproteinase-9 (MMP-9), TRAP and cathepsin K. Furthermore, the tea extract inhibited the activation of the intracellular signaling marker, NF-κB, at increasing concentrations.
Conclusion: This study demonstrates for the first time that rooibos tea may have potential anti-osteoclastogenic activity in vitro, which may indicate potential bone protective effects.
The study was supported by funding from RESCOM, UP and the IFNuW, UP
An in vitro study of newly designed and synthesized sirtuin 1 inhibitors on breast cancer cells
Nunes Gonçalves, J.P1
Department of Physiology, School of Medicine, Faculty of Health Sciences, University of Pretoria
Introduction: Sirtuin 1, a member of the Sirtuin family of regulatory deacetylase proteins has been shown to be upregulated in several cancers, including breast cancer. In women, breast cancer is the most common cancer diagnosed and the leading cause of cancer-related deaths. Inhibition of Sirtuin 1 expression has been shown to inhibit cancer growth in several previous publications. Two new in silico-designed, commercially unavailable Sirtuin 1 inhibitors have been previously created in our laboratory. Here, we use a breast cancer cell line model to test their effectivity.
Aim: To determine the effectiveness of the Sirtuin 1 inhibitors in vitro on metastatic and non-metastatic breast cancer cell lines MDA-MB-231 and MCF-7 respectively.
Methods: The effects of each of the new compounds, identified as BB1 and BB6, were determined via crystal violet assays. Light- and fluorescent microscopy were used to investigate cell morphology. Cell cycle and cell death analysis of treated cells were evaluated via flow cytometry. Molecular docking simulations were also done to determine the binding energies of each compound to Sirtuin 1 receptors.
Results: 50% inhibitory constants for the two inhibitors were determined to be 7,025 and 8,574 µM for BB1 and 2,582 and 2,873 µM for BB6 in MCF-7 and MDA-MB-231 cells respectively. Morphology studies revealed definite signs of cell damage and apoptosis in treated cells. Interestingly, no significant changes were noted in cell cycle analysis, however cell death studies showed significant increases in apoptotic cells, particularly in MDA-MB-231 cells.
Discussion: This study has shown that these newly designed and synthesized Sirtuin 1 inhibitors decrease cell growth in breast cancer cells at attractively low IC50’s. Although much research is still needed to confirm their effective use, in vitro results show promise as to the efficacy of the inhibitors in breast cancer, justifying further mechanistic studies.
The effect of nitric oxide donor treatment on skeletal muscle repair following contusion injury in rats
Prof Kathryn H Myburgh1
Introduction: Muscle injuries are highly prevalent and often lead to structural deficits such as fibrosis with subsequent functional deficits and recurrent injuries. Current treatment strategies have been shown to be ineffective and may even exacerbate damage (excess use of nonsteroidal anti-inflammatories). Nitric oxide (NO) is an endogenous bioactive molecule with several important physiological roles. NO knock-out models have shown inhibitory effects on regeneration, with excessive fibrosis. These observations lead to the aim of the current study: To clarify the anti-fibrotic role of NO following muscle trauma in a genomically unaltered model by treating with a NO donor or inhibitor; and to determine the effect that NO treatment or inhibition has on changes in physiological function following injury.
Methods: A contusion injury was performed by dropping a 250g weight on the gastrocnemius in rats, followed by treatment with placebo, Molsidomine (an NO-donor), L-NAME (a NO inhibitor), or a combination of NO and L-NAME. Treatments were administered immediately and one day post injury (n=6 in each group). Rats were sacrificed at 5 and 21 days post-injury. In situ mechanical force production was measured pre-injury and before sacrifice on each rat to determine changes in physiological function (Aurora Scientific, Ontario, Canada). Fibrosis was measured using Masson’s trichome and Sirius red staining. Staining with embryonic MHC was performed to identify new and regenerating muscle fibres.
Results: The force-frequency testing determined that maximal isometric force is exerted at 150Hz. Following injury, there was a significant reduction in maximal isometric force 5 days after injury (6.27±1.07 N/cm), compared to pre-injury (8.19±0.83 N/cm; p < 0.0001). At D21 there was a significant difference in maximal force between the Molsidomine group (8.31±1.19 N/cm) and both the L-NAME (7.39±1.46; p < 0.05) and combination (7.31±1.27 N/cm; p < 0.05) treatment groups.
Conclusion: Functional repair of the tissue occurs 21 days after injury as seen by recovery in maximal force production in placebo-treated rats. NO played a role in recovery of physiological function after a traumatic contusion as seen by the increase in force production at D21, compared to a reduction in maximal force following L-NAME treatment.
The effects of melatonin supplementation on vascular tissue during first line ART: an in vitro and in vivo study
Webster I,1Strijdom H,1Westcott C,1
Division of Medical Physiology, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Stellenbosch University1
Introduction: Cardiovascular disease (CVD) has emerged as a significant complication in HIV-infected patients, attributed in part to antiretroviral therapy (ART). ART is thought to impair endothelial function through increased reactive oxygen species (ROS) production. We aimed to assess the effects of melatonin (a potent antioxidant) in ART-treated rat aortic endothelial cells (AECs) and on the vascular reactivity of aortas obtained from rats treated with ART.
Methods: Different concentrations of melatonin and ART (EFV+FTC+TDF) were administered to serum-starved AECs either separately or as co-treatment for 1 or 24 h. Nitric oxide (NO), ROS and necrosis were measured in a platereader with appropriate fluorescent probes. Based on dose-response results, the co-treatment studies were performed with low-concentration melatonin (1 nM) and high-concentration ART (EFV: 5uM; FTC: 7.3uM; TDF: 1uM). Aortas were excised from male Wistar rats treated for 8-weeks with 10mg/kg/day melatonin and/or fixed-dose combination ART (EFV: 51.6mg/kg; FTC: 17.4mg/kg; TDF: 25.8mg/kg). Vascular reactivity was measured via aortic ring isometric tension studies.
Results: 1nM melatonin decreased necrosis after 1 h [Control PI: 100%±0.80; Mel PI: 81.89%±3.6;p<0.05] and 24 h [Control PI: 100%±1.041; 1 nM Mel PI: 92.56%±3.1; 1uM Mel PI: 100.9%±1.703; 10 uM Mel PI: 102.3%±2.075; p<0.05]. NO-production increased after 24 h treatment with high-concentration ART [Control DAF: 100%±2.224; high ART DAF: 112.7%±2.17; mid ART DAF: 100.8%±3.17; low ART DAF: 100%±2.7; p<0.05]. High-concentration ART also lead to increased ROS production [Control: 100%±0.79; ART: 108.8%±2.20;p<0.05]. In the co-treatment experiments, 1 nM melatonin + high-concentration ART decreased necrosis compared to ART alone [Control PI: 100%±1.067; ART+Mel PI: 94.17%±5.08; 1nM Mel PI: 92.43%±3.753; high ART PI: 121.3%±9.114; p<0.05]. In aortic ring experiments, the melatonin, ART and melatonin+ART groups all showed pro-contractile responses compared to untreated control [p<0.0001]. No differences in relaxation were observed.
Conclusion: ART treatment resulted in elevated levels of ROS and necrosis. Melatonin treatment was able to improve cell viability of normal control cells, as well as decrease ART-induced necrosis in vitro. In vivo treatment with melatonin did not show the same protective ability, only a pro-contractile effect. Signalling investigations will shed more light on this phenomenon.
Ex vivo apoptotic, autophagic and angiogenic influence of an estradiol analogue on platelets
Etheresia Pretorius1, Annie Margaretha Joubert1
Platelets are known contributors to vascularization, metastasis and growth of tumors. Upon their interaction with cancer cells they are activated resulting in the release of angiogenic activators thereby promoting angiogenesis. Angiogenesis-regulating proteins are ideal biomarkers in the study of cancer pathophysiology and represent desirable therapeutic- and diagnostic targets.
The in silico-designed analogue of 2-methoxyestradiol, namely 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16), binds to carbonic anhydrase II delaying early metabolism and is thus carried into the circulation. The effect it potentiates on blood components, especially on platelets, is of significance in cancer progression. This study thus investigated the possible ex vivo apoptotic, autophagic and angiogenic effects of ESE-16 on platelets.
Platelets were obtained from 3 healthy donors and exposed to physiological levels of ESE-16 as well as a known activation agent (4% DMSO), that acted as positive control for the experiments. Scanning electron microscopy was used to assess morphological changes in platelets after exposure to ESE-16 and no changes were observed in ESE-16-treated platelets. Experiments were repeated three times. The possible induction of apoptosis and autophagy was determined by annexin V-FITC, measurement of caspase 3 activity, autophagy-related protein 5 levels, light chain 3-I to light chain 3-II conversion and monodansylcadaverine staining which indicated that there was no increase in apoptosis or autophagy when platelets were exposed to ESE-16. The expression of the angiogenic proteins namely vascular endothelial growth factor, platelet derived growth factor and matrix metallopeptidase-9 was also assessed and results showed that levels were significantly increased after platelets were added to MCF-7 cells.
This is the first ex vivo study to highlight possible involvement of angiogenesis, apoptosis, autophagy in human platelets after exposure to this potential anti-cancer compound warranting further investigation concerning these signaling pathway targets on platelets of cancer patients.
Investigating the mechanisms of pharmacological chaperone rescue of mutant G protein-coupled receptors
Ross Anderson1, Robert Millar1, Craig Grobbelaar1
Centre for Neuroendocrinology, Department of Physiology1
Background: Dysregulation of G protein-coupled receptors (GPCRs) is commonly associated with a vast variety of diseases, including reproductive disorders associated with mutations found in the luteinizing hormone receptor (LHR). Many GPCR mutations cause protein misfolding and subsequent detection and retention by the ER quality control systems, preventing their expression at the cell surface. The ER quality control system has several levels of response to expression of misfolded proteins including the unfolded protein response (UPR) and ER associated degradation. Pharmacological chaperones, such as LHR-CHAP, have been shown to “rescue” the cell surface expression of retained mutant receptors. However, the mechanisms behind the “rescue” remain unclear and we therefore aimed to explore these processes in more detail.
Methods: An enzyme-linked immunosorbent assay, was employed to determine effects of treatment with LHR-CHAP for 24hrs on cell surface and total expression of a selection of mutant LHRs. Expression in the presence and absence of translation (Cycloheximide) or proteasome (MG132) inhibitors was also examined. Cellular localisation in the presence/absence of LHR-CHAP was addressed through confocal microscopy by co-transfecting cells with fluorescently tagged wild type/mutant receptors and markers of different cellular compartments. The expression profiles of molecular chaperones involved in the UPR were investigated using a luciferase reporter gene assay and Western blotting
Results: Localisation studies demonstrated that wild type LHR is expressed on the cell membrane (as expected), whereas mutant LHRs co-localize with different cellular compartments involved in the folding and maturation of secretory receptors, and that treatment altered localisation of the retained receptors. LHR-CHAP upregulated the expression of various components of the UPR, but again, differences were observed for the different mutant receptors. Inhibition of newly synthesised receptors didn’t affect the “rescue” response of LHR-CHAP, however it was found that the inhibition of the proteasome interferes with trafficking of mutant receptors to the cell surface, suggesting a role for the proteasome in processing of these receptors.
Conclusions: Different mutant LHRs are differentially processed by the cell and are retained and rescued to different degrees. Pharmacological chaperones, such as LHR-CHAP, show potential as drug therapies to rescue mutant LHRs associated with diseases such as infertility.
Effect of Melodene use on whole blood clot formation and erythrocyte morphology of a healthy female: a case study
BarendGerhardus Lindeque1, Albe Carina Swanepoel1
University of Pretoria1
Introduction: The use of synthetic steroid hormones for oral contraceptive purposes has a well-established risk of leading to venous thromboemboli (VTE). Venous thrombi are commonly referred to as “red-clots” due to the thrombus composition consisting mainly of erythrocytes and fibrin. This case study investigates the possible alterations in viscoelastic parameters of whole blood clots as well as the biophysical and biochemical characteristics of erythrocytes in a female currently on the combined oral contraceptives (COC) Melodene.
Methods: A whole blood sample from a healthy female volunteer currently using Melodene was analysed with regard to three parameters. These parameters focus on the effects that Melodene use have on erythrocytes.
Whole blood counts of the participant were performed by means of a haematology analyser. This provided an overview of the blood profile. Erythrocyte sedimentation rate (ESR) was done to determine the level of inflammation of the sample. Thromboelastography (TEG) was used to measure several parameters surrounding clot formation of whole blood and thus determine the viscoelastic properties of the whole blood sample. The impact on the biophysical characteristics of erythrocytes specifically the morphology and membrane ultrastructure was analysed using scanning electron microscopy.
Results: The overall blood profile as determined by the whole blood counts indicated that the participant was in general good health. An ESR value of 3mm.h-1 falls well within the normal range of 0-29mm.h-1 indicating no abnormal inflammatory state. Viscoelastic measurements indicated that only the reaction was influenced; it was decreased compared to the normal reference ranges for WB TEG analysis while all other parameters were within the normal reference ranges. Erythrocyte ultrastructure remained largely intact. Spontaneous platelet activation was observed throughout the sample. A pronounced increase in rouleaux formation was also observed as well as a continuous fibrin layer enveloping the erythrocytes.
Conclusion: This case study indicates that Melodene usage had a very subtle effect on coagulation as determined with quantitative tests. The most striking effects were observed with regard to the ultrastructural analysis indicating a continuous fibrin layer enveloping the erythrocytes as well as spontaneous platelet activation and increased rouleaux formation.
Modulation of osteoblast differentiation and function by aspalathin and gallic acid found in Aspalathuslinearis (Rooibos)
Abe Kasonga1, Ulrike Baschant1, Prof Marlena Kruger2
Department of Physiology, Faculty of Health Sciences, University of Pretoria1
Massey Institute of Food Science and Technology, Massey University, Palmerston North, New Zealand2
Bone remodeling in the skeleton is an ongoing process that entails constant balance between bone resorption by osteoclasts and bone formation by osteoblasts. Diseases in which bone remodeling is not in balance, such as osteoporosis and rheumatoid arthritis, are highly prevalent in a modern world. Previous studies have shown that tea made from Aspalathuslinearis (Rooibos) may have bone protective effects by reducing osteoclast differentiation in vitro. Aspalathin and gallic acid are compounds found in rooibos that show promising effects on systems implicated in bone remodeling. This study aimed to test the effects of aspalathin and gallic acid on the differentiation and function of osteoblasts in vitro.
Osteoblast work was conducted using primary extracted mouse bone marrow cells. Celltiter Blue cytotoxicity assay was used to measure the effects of compounds on bone marrow cell viability. Alizarin Red staining and alkaline phosphatase expression were measured to test the effects of compounds on osteoblast differentiation. Tartrate-resistant acid phosphatase staining was used to study the effects of the compounds on an osteoblast-osteoclast co-culture. qRT-PCR was used to determine the effects of the compounds on osteoblast marker gene expression.
Celltiter Blue assays showed that aspalathin had no significant effect on cell viability up to 1000 µM, and gallic acid up to 100 µM. Alizarin Red S and alkaline phosphatase experiments showed that aspalathin increased osteoblast mineralization at 500 µM and gallic acid at 50 µM. In primary osteoblast-osteoclast co-cultures aspalathin and gallic acid reduced osteoclast differentiation at 500 µM and 50 µM, respectively. qRT-PCR experiments showed that aspalathin (500 µM) increased Runx2, BMP-2, and osterix expression, while decreasing SOST expression. Gallic acid (50 µM) increased Osterix, BMP-2 and Collagen 1A expression, while decreasing SOST expression.
Both aspalathin and gallic acid show potential bone protective effects in vitro as they increased mineralization and the expression of osteoblast differentiation markers. Both compounds reduced the expression of the osteoblast inhibition marker SOST. In osteoblast-osteoclast co-cultures, both compounds reduced osteoclast differentiation. This study shows that these compounds may increase osteoblast differentiation and function, while reducing osteoclast differentiation in vitro.
Microtubule disruption contributes to the pro-apoptotic effect of steroidomimetictetrahydroisoquinolinone-based analogues in cancer cells
Anna M Joubert1, Wolfgang Dohle2, Barry VL Potter2
Department of Physiology, Faculty of Health Sciences, University of Pretoria1
Medicinal Chemistry & Drug Discovery, Department of Pharmacology, University of Oxford, Mansfield Rd, Oxford OX1 3QT, United Kingdom2
Introduction: Tetrahydroisoquinolinone (THIQ)-based analogues were synthesized by incorporating the essential pharmacophore elements of steroidal microtubule disrupters namely 2-methoxyestradiol and 2-methoxyestradiol-3,17-O,O-bis-sulphamate (STX 140), into the non-steroidal THIQ core in order to better the parent compounds’ pharmaceutical properties. The aim of this in vitro study was to investigate the novel chimeric THIQ-based analogues (STX 2895, STX 3329, STX 3450 and STX 3451) ability to exert anti-proliferative- and anti-mitotic effects culminating in the induction of apoptosis in both MDA-MB-231 and A549 neoplastic cell lines.
Methods: The half maximal growth inhibitory concentration (GI50) concentrations were determined with spectrophotometry. Microtubule integrity, acidic vesicle formation and morphologic changes of treated cells were investigated using light microscopy, transmission electron microscopy and fluorescent microscopy. Signalling pathways for apoptosis induction were determined by quantification of phosphatidylserine flip, mitochondrial membrane depolarization, cytochrome c release and cell cycle progression analysis using flow cytometry.
Results: Cytotoxicity studies determined the GI50 values of the analogues to be at nanomolar concentrations, without the induction of necrosis. Their anti-mitotic effect was demonstrated through confocal microscopy which demonstrated abrogated microtubule networks. Furthermore, a clear G2/M block was quantified through analysis of the cell cycle which was corroborated by the presence of rounded cells observed in light microscopy. Mitotic arrest induced apoptotic cell death, a finding supported by an elevated sub-G1 population as well as an increased phosphatidylserine flip in treated cells. Light– and transmission electron microscopy revealed the presence of shrunken cells, hypercondensed chromatin and apoptotic body formation. Mitochondrial membrane potential reduction as well as the increased release of cytochrome c indicated compound exposure for 24 hours induced the intrinsic apoptotic pathway. Potential involvement of programmed cell death type II became evident due to the increase in acidic vacuoles and aggresome formation.
Conclusion: This in vitro study revealed that the novel chimeric THIQ-based analogues exert cytotoxic and anti-mitotic effects at nanomolar concentrations culminating in the induction of the intrinsic apoptotic pathway in both MDA-MB-231 and A549 cell lines. These results provide a basis for future in vitro and in vivo investigations to develop the newly synthesized analogues into a clinically usable anticancer drugs.
Electrical and cardiac stress responses associations with sub-clinical target organ damage: The SABPA study
LeonéMalan1,Roland VON Känel1,2,3, Shani Botha1,Nicolaas T Malan1
Hypertension in Africa Research Team (HART), School for Physiology, Nutrition, and Consumer Science, North-West University, Potchefstroom1
Department of Psychosomatic Medicine, Clinic Barmelweid CH-5017, Barmelweid, Switzerland2
Department of Neurology, Inselspital, Bern University Hospital, and University of Bern, Switzerland3
Autonomic responsivity of the heart is a recognized as a risk factor for cardiovascular disease (CVD). However, the manner in which cardiac stress markers, i.e. cardiac troponin T (cTnT) and N-terminal pro-brain natriuretic peptide (NT-proBNP) levels, change in response to acute mental stress, and whether these levels relate to specific vascular and-cardiac responses, remains unclear. Africans (n=193) and Caucasians (n=195) from the Sympathetic activity and Ambulatory Blood Pressure in Africans (SABPA) study, were included in our analysis. The Colour-Word Conflict (STROOP) test was administered for 1 minute whilst Finapres® beat-to-beat BP and 10-lead ECG responses were obtained. Blood samples for cTnT, NT-proBNP and cardio-metabolic markers were obtained at baseline and 10 min post-stress application. CVD markers changed detrimentally in Africans who exhibited greater vascular, cTnT and NT-proBNP responses compared to Caucasians.
There were inverse associations of stroke volume with ∆DBP (β=-0.31; 95%CI -0.38 to -0.20; p<0.001), ∆cTnT (β=-0.13; CI -0.22 to -0.01; p=0.009) and ∆QTc (β=-0.25; CI -0.35 to -0.15; p<0.001) in Africans. ROC analyses revealed both that acute increases (4.16pg/mL) in and resting values (4.19pg/mL) of cTnT were associated with 24 hour diastolic hypertension [specificity/sensitivity 65%/69%; AUC 0.63 (95% CI, 0.55, 0.70)] in Africans. The R wave of the aVL lead predicted acute stress induced and resting increases in cTnT [Odds ratio of 11 (95%CI)] in the Africans only – indicating a possible maladaptive response to acute and everyday stress. CVD vulnerability was accompanied by acute cardiac stress and α-adrenergic responsivity in Africans. Ultimately, this reaction may increase the risk for ischemic heart disease.
Key words: Stressor responses; ethnicity; South Africa; Cardiac Troponin T; NT-proBNP; CVD