Masters Degrees (Medical Biochemistry)

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1,4,7,10,13,16-Hexaazacyclooctadecane (Hexacyclen) Induced Nitrosative Stress and Downregulated NF-κB Cell Survival Pathway in Human Embryonic Kidney (Hek293) and Colorectal Adenocarcinoma (Caco2) Cells.
(2022) Nxumalo, Mthokozisi Bongani.; Khan, Rene Bernadette.; Khumalo, H.
Colorectal cancer (CRC) is the third most common malignancy detected and the second leading cause of cancer-related mortality. Mammalian cells require metals for the physiological process as they are part of the structure or co-factor of many proteins. However, excessive accumulation may manifest in toxicity. In addition, the promotion of oncogenesis and tumour growth has been associated with an increased presence of metals. Promising anticancer compounds that disrupt the onset and progression of carcinogenesis are currently being intensely investigated by the scientific community. Hexacyclen, a nitrogen electron donor and a potent metal ion chelator that binds various metal and transition metal cations, is one such anticancer drug. The cytotoxic effects of Hexacyclen on human colorectal adenocarcinoma cells (Caco2) and normal embryonic kidney cells (Hek293) were investigated in this work after acute exposure (48 hours). The toxicity of Hexacyclen was studied in Hek293 and Caco2 cells at different concentration ranges [(0-500 μM) and (0-50 μM), respectively]. The MTT (to determine IC20 and IC50), ATP and mitochondrial membrane potential (ΔΨM) assays were used to assess metabolic activity, while TBARS, NOS and GSH assays were used to assess oxidative activity. Caspase activity (-8, -9, -3/7), phosphatidylserine externalisation and LDH leakage were used to assess cell death by apoptosis. In addition, western blotting was used to examine the expression of antioxidant (SOD2, GPx, catalase), pro-and anti-apoptotic (p-p53, Bcl-2, HSP70, PARP, cPARP) and inflammatory (NF-κB, STAT3 and p-STAT3) proteins. From the dose-dependent MTT curve, an IC20 and IC50 of 6μM and 38μM (Hek293) and 1.2μM and 5μM (Caco2 cells) were determined. The decreased ATP concentration in Hek293 (p<0.05) and Caco2 (p>0.05) cells for both treatments was consistent with altered ΔΨM in both cell lines, indicating reduced metabolic activity. Elevated RNS was implied by increased iNOS particularly at the Caco2 IC50 (p<0.05) that promoted nitric oxide production at the IC20 (p>0.05) and IC50 (p<0.05) for Hek293 and Caco2 cells respectively. The decreased MDA in Hek293 cells (p>0.05) was associated with increased SOD2 (p<0.05) and GPx (p<0.05), while slightly increased MDA in Caco2 cells (p>0.05) accompanied increased SOD2 (p>0.05) and GPx (p<0.05 at the IC50 only). Furthermore, GSH levels were increased significantly in IC50-treated Hek293 and Caco2 cells (p<0.05), but downregulation of catalase in Hek293 and Caco2 cells was not significant. In this study, apoptosis was initiated by an increase in caspase-9 (IC50, p<0.05) but not caspase 8, which was decreased for both treatments in Hek293 cells (p<0.05). In Caco2 cells, caspase-8 (p<0.05) and caspase 9 (p>0.05) were increased. Anti-apoptotic Bcl-2 (p<0.05) and HSP70 (p<0.05 for Caco2 cells) were downregulated in both cell lines. The activity of p-p53 was not affected in IC20, whereas it was significantly reduced in IC50-treated (p<0.05) in Hek293 and Caco2 cells. Apoptosis was executed as caspase 3/7 was increased in all treatments (p<0.05), albeit non-significantly for IC20-treated Hek293 cells. Moreover, phosphatidylserine externalisation, an early apoptosis marker, was increased in both cell lines (p<0.05 for IC50-treated Hek293 cells), while LDH (a late marker) was increased for Hek293 cells (p<0.05) but not Caco2 cells (p>0.05). Interestingly, decreased cPARP/PARP activity was observed for IC50-treated cells (p<0.05) in both cell lines. Finally, the inflammatory markers NF-κB (p>0.05 for IC20-treated Hek293 cells) and p-STAT3/STAT3 (p>0.05 for IC20-treated Caco2 cells) were downregulated in this study. Hexacyclen induced apoptosis in Hek293 and Caco2 cells via an RNS-mediated mechanism. Intrinsic apoptosis was noted in Hek293 cells, while both pathways facilitated apoptosis in Caco2 cells. Interestingly, apoptosis proceeded concurrently with a reduction in the NF-κB cell survival pathway.
Aflatoxin B1 modulates oxidative stress and apoptosis in human embryonic kidney cells.
(2019) Dlamini, Nomali Zanele.; Khan, Rene Bernadette.
Introduction: Aflatoxin B1 (AFB1) is produced by filamentous fungal strains of Aspergillus flavus and Aspergillus parasiticus that infect field crops, therefore AFB1 is a frequent contaminant of dietary staples such as rice, maize and peanuts. Humans and animals are exposed to AFB1 through consumption of contaminated foods, predisposing them to various diseases. AFB1 is a potent hepatotoxin that has been classified by the International Agency of Research on cancer (IARC) as a group1 carcinogen. The carcinogenic effects of AFB1 have been attributed to the metabolism of this toxin to an epoxide that promotes the production of free radicals, mitochondrial toxicity and induction of cell death. With the increasing prevalence of kidney associated diseases in humans, and the AFB1-associated kidney toxicity observed in animals, this study investigated the cytotoxic effects/mechanism of AFB1 in human embryonic kidney (Hek293) cells. Methods: Hek293 cells were exposed to AFB1 (0-100μM) for 24hrs. The effect on cell viability was assessed using the methylthiazol tetrazolium (MTT) assay, which also produced the half maximal inhibitory concentration (IC50) used in subsequent assays. Free radical production was evaluated by quantifying malondialdehyde (MDA) and nitrate concentration, while DNA fragmentation was determined using the single cell gel electrophoresis (SCGE) assay and DNA gel electrophoresis. Damage to cell membranes was ascertained using the lactate dehydrogenase (LDH) assay. The concentration of ATP, reduced glutathione (GSH), necrosis, annexin V and caspase activity was measured by luminometry. Western blotting and quantitative PCR was used to assess the expression of proteins and genes associated with apoptosis and oxidative stress. Results and discussion: The MTT assay revealed a reduction in cell viability of Hek293 cells as the AFB1 concentration was increased, with a half maximum inhibitory concentration (IC50) of 32.60 μM. The decreased viability corresponded to decreased ATP concentration. The upregulation of Hsp70 indicated that oxidative stress was induced in the AFB1-treated cells. While this implies an increased production of free radicals, the accompanying upregulation of the antioxidant system indicates the activation of defense mechanisms to prevent cellular damage. Thus, membrane damage associated with increased radical formation was prevented as indicated by the reduced LDH release and necrosis. In addition, cytotoxic effects were evident as AFB1 activated the intrinsic pathway of apoptosis with corresponding increased DNA fragmentation, p53 and Bax upregulation and increased caspase activity, but externalisation of phosphatidylserine (PS), a major hallmark of apoptosis, did not occur in AFB1 treated Hek293 cells. Conclusion: The results suggest that AFB1 induced oxidative stress leading to cell death by the intrinsic pathway of apoptosis in Hek293 cells. Keywords : Aflatoxin B1 (AFB1), oxidative stress, apoptosis, Hek293 cells
Allicin ameliorates some deoxynivalenol-induced cytotoxic effects in human embryonic kidney (Hek293) cells, but also elicits synergistic and potentiating adverse effects.
(2020) Mamane, Yandisa Zintle.; Khan, Rene Bernadette.
Introduction: Deoxynivalenol (DON), a type B trichothecene produced by plant pathogenic fungi, especially Fusarium graminearum and F. culmorum, is a highly toxic mycotoxin found throughout South Africa. DON is consumed unintentionally through maize derived products and is rapidly becoming a potential health risk to humans and animals. It is a known immunosuppressant that induces apoptosis and oxidative stress and may cause liver lesions and kidney problems. Recently, dietary therapeutics have demonstrated a role against mycotoxin-induced cytotoxicity. Garlic (Allium sativum) is part of the Alliaceae family. The garlic bulb is used for medicine and as food consumption. The aqueous extract has recently demonstrated the potential to protect against mycotoxin-induced cell death and decrease reactive oxygen species (ROS). Aim: This study investigated the induction of apoptosis and oxidative stress by DON in Hek293 cells, and the ability of allicin to ameliorate these effects. Methods: Hek293 cells were treated with a range of allicin concentrations (0-150mM) over 24hrs. An EC50 of 1.7mM was obtained from the MTT assay and used in all subsequent assays. Hek293 cells were treated with 5μM DON, 1.7mM allicin (A), or a combination (DON+A) for 24hrs; untreated cells served as the control. Lipid peroxidation [malondialdehyde (MDA) and lactate dehydrogenase (LDH) assays] were used to indirectly quantify reactive oxygen species (ROS) and oxidative stress; reactive nitrogen species (RNS) were quantified using the nitrates assay. Apoptotic induction was determined by the detection of phosphatidylserine (annexin V) and DNA fragmentation. Necrotic cells were distinguished by propidium iodide uptake. Luminometric quantification of ATP, reduced glutathione (GSH), and caspase 9, 3/7, were used to verify these events. In addition, antioxidant enzymes protein expression of superoxide dismutase (SOD2), catalase and glutathione peroxidase (GPx1); as well as nuclear factor erythroid 2-related factor 2 (Nrf2) and heat shock protein (Hsp70), and apoptotic markers associated protein expression of p53, Bax, and poly (ADP-ribose) polymerase (PARP) were detected by western blotting. Results: DON-induced ROS production was suggested by the depletion of antioxidants including SOD2 (p < 0.0001), catalase (p < 0.0001) and GSH (p = 0.0886). Decreased lipid peroxidation indicated by the decreased MDA concentration (p < 0.0001) and reduced LDH (p = 0.0342) imply that the Hek293 cells were spared from the membrane-damaging effect of oxidative stress. A reduction in Hsp70 (p = 0.0056) and Nrf2 (p < 0.0001), and upregulation of GPx1 (p = 0.0362) protein expression was noted. In addition, increased nitrate concentration in all treatments compared to the control (p < 0.0001) suggested a shift to RNS production. Notably, allicin maintained Nrf2 protein expression similar to the control. The decrease in MDA concentration (p = 0.0109) by allicin was concurrent with depleted GSH (p = 0.0504)and increased SOD2, catalase and GPx1 (p < 0.0001), and suggests allicin induced an oxidative stress response. Allicin also protected DON-treated cells from oxidative stress by upregulating Hsp70 (p < 0.0001), catalase (p = 0.0006) and GPx1 (p = 0.0018), with concurrent decreased GSH (p = 0.0342) and ATP (p = 0.2028) concentration, which were also decreased by DON. In addition, allicin increased MDA (p < 0.0001) and LDH (p = 0.1267) towards control levels in the combined treatment. Apoptosis was reduced in the DON (p = 0.4631) and DON+A (p < 0.0488) treated cells in comparison to the control, necrosis was not evident in any treatment. The slight induction of p53 (p = 0.0008) and PARP-1 (p = 0.4036) by DON implies an attempt at DNA repair, but the Hek293 cells experienced reduced levels of apoptosis. Indeed, Bax expression was slightly reduced (p = 0.1071), caspases 9 (p = 0.0705) and 3/7 (p = 0.4431) activities were diminished, phosphatidylserine was not externalized, and PARP-1 was not cleaved. A non-fragmented DNA profile in allicin-treated and DON+A-treated Hek293 cells may be explained by increased expression of DNA repair proteins, PARP-1 (p = 0.0048 and p = 0.0004 respectively) and p53 (p < 0.0001). The upregulation of p53 is associated with an increase in Bax expression (p < 0.0001 and p = 0.0026 respectively). However, caspases 9 (p = 0.0596) and 3/7 (p = 0.0311) were not activated and apoptosis did not occur. Conclusion: DON treatment induced oxidative stress but not apoptosis in Hek293 cells at the concentration tested. In addition, its mechanism of toxicity in Hek293 cells appears to be more related to nitrosative stress and induction of DNA damage. Oxidative stress and not apoptosis is the possible mechanism of allicin-induced effects in Hek293 cells. Although allicin ameliorated some of the effects of DON in Hek293 cells, it also elicited synergistically or potentiating adverse effects that require further investigation.
Analysis of a multidrug resistant acinetobacter SPP. outbreak in the intensive care unit of King Edward VIII Hospital.
(2000) Deedat, Fathima.; Sturm, Adriaan Willem.
The study arose out of a need to investigate and control a nosocomial outbreak caused by multidrug resistant Acinetobacter spp in the fifteen-bed intensive care unit of King Edward VIII Hospital. Following the discovery of the index case, four other patients were found to have a similar strain of Acinetobacter spp. All fifteen patients in the ward were subsequently screened for the organism. Forty-seven isolates were obtained from 12 patients. Eight of the patients were infected with the organism and six of these eight patients subsequently died. Swabs from the ward environment were also screened for the organism, which was found in patients' baths, suction water and urine collection jars. The outbreak was aborted by the use of strict infection control techniques. Minimum inhibitory concentrations (MICs) of 20 of the 47 isolates were determined for the following antimicrobials: imipenem, ciprofloxacin, gentamicin, amikacin, netilmycin,cefotaxime, ceftazidime and tetracycline. The same 20 isolates were further typed using ribotyping. Seven different antibiogram patterns were obtained using the MIC data. The majority of isolates (11) fit into a Single type, and showed resistance to all drugs tested, except for susceptibility to tetracycline and netilmycin only. Ribotyping revealed 5 different types. There were 9 isolates of ribotype a, 2 of ribotype b, 3 of ribotype c, 5 of ribotype d and 1 of ribotype e. In conclusion, this study describes a nosocomial outbreak with a multidrug resistant Acinetobacter spp. in an intensive care unit. The results showed that there was no correlation between the two typing methods used, ribotyping was more discriminatory than antibiogram types, with the majority of strains belonging to two different ribotypes.
Antiproliferative effect of a novel synthesized carbazole compound on A549 lung cancer cell line.
(2014) Molatlhegi, Refilwe P.; Chuturgoon, Anil Amichund.
Increased death rates due to lung cancer have necessitated the search for potential novel anticancer compounds such as carbazole derivatives. Carbazoles are aromatic heterocyclic compounds with anticancer, antibacterial and anti-inflammatory activity. The study investigated the ability of the novel carbazole compound (Z)-4-[9-ethyl-9aH-carbazol-3-yl) amino] pent-3-en-2-one (ECAP) to inhibit the proliferation of lung cancer cells and its mechanism of action. ECAP was synthesized as a yellow powder with melting point of 240-247 °C. The 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), lipid peroxidation and comet assays were used to assess the anti-proliferative effects of the compound on A549 lung cancer cell line. Protein expression was determined using western blots, apoptosis was measured by luminometry for caspase-3/7, -8 and -9 and flow cytometry was used to measure phosphatidylserine externalisation. ECAP induced a p53 mediated apoptosis of lung cancer cells by significantly down-regulating the expression of antioxidant defense proteins, Hsp70 (p < 0.02) and Bcl-2 (p < 0.0006), thereby up-regulating reactive oxygen species (ROS) production. This resulted in DNA damage (p < 0.0001) and subsequent up-regulation of Bax and caspase activity consequently inducing apoptosis of lung cancer cells. These results demonstrate the potential anticancer effects of ECAP on cultured lung cancer cells. However, further investigation and characterization is required to fully understand the possible use of carbazole compound (Z)-4-[9-ethyl-9aH-carbazol-3-yl) amino] pent-3- en-2-one as potential lung cancer treatment.
Artemisia afra crude aqueous leaf extract indices oxidative stress and inflammation in human colon adenocarcinoma cells via the upregulation of the TNF-a,p38 and STAT3 pathway.
(2022) Mposula, Slindelo.; Khan, Rene Bernadette.
ABSTRACT Introduction: Artemisia afra (A. afra) is a widely used medicinal plant located in the southern African region. It is traditionally used to alleviate medical conditions such as coughs. Literature indicates a protective role by improving antioxidant capacity and reducing cell proliferation, which suggests anti-cancer potential. Colorectal carcinoma (CRC) is a global public health crisis and the second common cause of cancer-related fatalities. Current cancer treatment is deemed effective but not easily accessible and expensive in the southern African region. Therefore, the need for naturally derived anti-cancer agents remains to be investigated for accessible and affordable treatment. This study investigates the antiproliferative and antioxidant effects of A. afra crude aqueous leaf extract in the Caco-2 cell line. Materials and Methods: Caco-2 cells were treated with a range of A. afra concentrations (0-5000 μg/ml) for 48 hours. An IC50 was derived from the MTT assay and all subsequent assays compared the IC50 -treatement to an untreated control. Mitochondrial integrity was luminometrically assessed by measuring JC-10 fluorescence and ATP. Free radical production (TBARS, NOS) and membrane damage (LDH cytotoxicity), together with GSH quantitation were used to infer the presence of oxidative stress; antioxidant enzymes (SOD2, GPx-1, catalase, Nrf2) were also detected by western blotting. Apoptotic induction was verified by measuring phosphatidylserine externalisation, quantifying caspase activities and detecting pro- and anti-apoptotic proteins (Bax, Bcl2, cIAP, xIAP) by western blotting. Single strand DNA fragmentation was evaluated via the comet assay. Additionally, relative expression of DNA repair, inflammation and stress markers were determined using western blotting and qPCR. Results: Crude aqueous leaf extract of A. afra induced a dose-dependent reduction in cell viability, yielding an IC50 of 250 μg/ml. Decreased mitochondrial integrity (p = 0.697) was associated with significant depletion of intracellular ATP (p = 0.0043) and increased ROS production as validated by increased lipid peroxidation (p = 0.1638) and DNA oxidation (amplified OGG1). In addition, increased iNOS contributed to the production of RNS. Artemisia afra induced an antioxidant response that elevated Nrf2 at the mRNA and protein level, causing increased GSH (p = 0.0001), GPx-1 (p = 0.5067) and catalase, but SOD2 was decreased. Heightened levels of heatshock proteins (HSP27 and HSP70) correlate with increased ROS and upregulated phosphorylated p38 protein, but ERK and JNK protein expression was downregulated. Significant downregulation caspase-8 (p = 0.0252), caspase-9 (p = 0.0099) and caspases-3/7 (p = 0.0232) was associated with reduced Annexin-V) and extracellular LDH. In addition, the Bax/Bcl-2 ratio (p = 0.0033) and protein expression of inhibitors of apoptosis protein such as cIAP-1 and xIAP indicated reduced apoptotic activity in this study. Comet tail analysis indicated intact DNA, in congruence with decreased OGG1. Both TNF-α (p = 0.2323) and STAT-3 were upregulated, but NF-ĸB was decreased. In addition, cellular Myc and phosphorylated retinoblastoma were upregulated. Conclusion: The crude aqueous leaf extract of A. afra induced mitochondrial toxicity and ROS production. Despite a heightened antioxidant defense, ROS-mediated upregulation of TNF-, p38 and STAT3 promoted cell proliferation and inhibited apoptosis in Caco-2 cells. Taken together, A. afra is a cytotoxic and genotoxic agent that may induce cancer in human colorectal cells.
Atorvastatin induces apoptosis via the P13K/AKT signalling pathway in human hepatocellular carcinoma (HEPG2) cells.
(2016) Docrat, Taskeen Fathima.; Chuturgoon, Anil Amichund.
Abstract available in PDF file.
Betulinic acid enhances the antioxidant profile in a hyperglycaemic model.
(2019) Maharaj, Gopala.; Chuturgoon, Anil Amichund.; Sheik-Abdul, Naeem.
Type 2 diabetes mellitus (T2DM) is a global pandemic, with prevalence rapidly rising in South Africa. T2DM is characterized by insulin resistance, leading to hyperglycaemia which induces oxidative stress (OS) and inflammation with subsequent complications. Betulinic acid (BA), a ubiquitous plant triterpenoid, has many proven benefits including antioxidant (AO) properties. Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor which binds to triterpenes and promotes glucose uptake and stimulates cytoprotective and anti-inflammatory effects. This study investigated the potential of BA to modulate cytoprotective responses through PPARγ in response to hyperglycaemic (HG) induced OS in a human hepatoma (HepG2) liver cell model. HepG2 cells were cultured under normoglycaemic (NG) and HG conditions and subsequently treated with 5μM and 10μM BA. Spectrophotometric [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays] and luminescent (ATP assay) principles were employed to assess viability of the chosen BA concentrations. Phosphorylation of the insulin receptor β-subunit (IRβ) was assessed via Western blot to confirm BA’s anti-HG effects. Intracellular reactive oxygen species (ROS) levels were assessed via fluorescence using the 2′,7′-dichlorodihydrofluorescein-diacetate (H2DCF-DA) assay, and oxidative stress biomarkers were quantified spectrophotometrically, via use of the thiobarbituric acid reactive substances (TBARS) assay for lipid peroxidation, and protein carbonyl assay (PCA). Intracellular AO potential was measured via luminometric quantification of reduced glutathione (GSH). Western blots quantifying protein expression of PPARγ, nuclear factor erythroid 2-related factor2 (NRF2), phosphorylated NRF2 (pNRF2), sirtuin3 (SIRT3), PPARγ coactivator 1α (PGC1α), superoxide dismutase 2 (SOD2), catalase (CAT), uncoupling protein 2 (UCP2), lon protease (LONP1) and nuclear factor κ-B (NFκB) as well as quantitative polymerase chain reaction (qPCRs) assessing gene expression of glutathione peroxidase (GPx1), NRF2, SIRT3, PGC1α and micro-RNA 124 (miR124) were run to elucidate the molecular mechanism behind the cytoprotective response of BA. The MTT, ATP and LDH assays confirmed cell viability, lack of toxicity and stable energy output, while TBARS, DCF and PCA confirmed a reduction of ROS and its biomarkers. A preliminary Western blot of IRβ confirmed BA’s anti-hyperglycaemic actions at a prime concentration of 5μM BA. Further, Western blots also confirmed an AO-induced protective mechanism at 5μM BA originating from the PPARγ/NRF2 positive feedback loop, further involving SIRT3 (p<0.0001), PGC1α (p=0.0025), LONP1 (p<0.0001), and AOs: SOD2 (p<0.0001), CAT (p=0.0003) and UCP2 (p<0.0001). The GSH assay and mRNA levels of PGC1α (p<0.0001), NRF2 (p<0.0001), SIRT3 (p<0.0001) and GPx1 (p<0.0001) further confirmed the mechanism, while miR124 levels (p=0.0093) hinted at epigenetic regulation between the transcription factors. Additionally, BA was found to downregulate NFκB (p<0.0001) in the HG state possibly combatting ROS-induced inflammation. In conclusion, BA illustrated cytoprotective effects on HG induced OS at an optimum concentration of 5μM, by upregulating the AO response and reducing ROS. Thus, BA may be considered an alternate and cheap adjunctive therapy to mitigate complications of T2DM.
Biotyping Saccharomyces cerevisiae strains using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS)
(2011) Moothoo-Padayachie, Anushka.; Kruger, Hendrik Gerhardus.; Maguire, Glenn Eamonn Mitchel.; Govender, Thavendran.; Govender, Patrick.
In clinical diagnosis and fermentation industries there is a need for a method that allows for the differentiation of yeast to the strain level (biotyping). The ideal biotyping method should be accurate, simple, rapid and cost-effective, and capable of testing a large number of yeast isolates. Matrix assisted laser desorption/ionization time of flight mass spectrometry has emerged as a powerful biotyping tool for the identification of bacteria and clinical yeast isolates, mainly Candida. It has been found that the MALDI-TOF MS signals from yeast are harder to obtain than from bacteria. It has been reported by several research studies that a cell lysis step is required to obtain a mass spectral signal for clinical Candida strains. To date an optimized sample preparation protocol has not been devised for the biotyping of S. cerevisiae strains. Studies on the identification of yeast using MALDI-TOF MS have focused primarily on clinical Candida yeast isolates but have included very few S. cerevisiae strains. Furthermore these yeast identification studies using MALDI-TOF MS have only achieved identification to the species and not strain level. A major limiting attribute of MALDI-TOF MS for the accurate identification of microbes, is its dependency on a comprehensive mass spectral database. Bruker Daltonics is a pioneer and leader in providing innovative life science tools based on mass spectrometry thus the Bruker Daltonics mass spectral database and state-of-the-art instruments and accompanying software were selected for this study. The Bruker Daltonics mass spectral database currently holds three thousand seven hundred and forty microorganisms of which only a mere seven are S. cerevisiae strains. Initially in this study, a number of parameters of a generic ethanol/formic acid protein extraction procedure as originally described by Bruker Daltoincs were considered in the development of a sample preparation protocol that yielded characteristic and highly reproducible MALDI-TOF mass spectra. The parameters considered included cell number, alcohol fixation, matrix solution and media. It was found that using the optimized sample preparation protocol unique and highly reproducible mass spectral profiles were obtained for all three S. cerevisiae strains. Multivariate analysis confirmed that the differences between all three S. cerevisiae strains were statistically significant. For quality assurance, the spectra of the three strains were sent for evaluation by Bruker Daltonics and were deemed suitable for the purpose of biotyping. The newly created ethanol/formic acid extraction procedure was used to generate an S. cerevisiae mass spectral database comprising of forty-five S. cerevisiae strains within a local context but also of global significance. The accuracy of the mass spectral database was assessed using blind coded S. cerevisiae strains obtained from the Agricultural Research Council Infruitec-Nietvoorbij (Institute for Deciduous Fruit, Vines and Wine), Stellenbosch, South Africa. It was found that S. cerevisiae identification to the species and more importantly strain level was achievable with relatively good accuracy. To determine the potential application of MALDI-TOF MS as an accurate method for S. cerevisiae strain identification in industry, blind coded S. cerevisiae strains were obtained from Natal Cane Products and subjected to MALDITOF MS analysis. It was found that four of the pure cultures submitted were correctly identified to the strain level and the three S. cerevisiae strains incorrectly identified may have been contaminants or the result of incorrect optimization conditions for the fermentation. Thus MALDITOF MS was shown to be an accurate identification tool, that may also be used to detect contaminants or incorrect environmental conditions which can result in substantial losses.
Characterisation of Fumonisin B1 toxicity in a cancerous liver cell line- induction of tissue transglutaminase and the endoplasmic reticulum stress pathway.
(2013) Anderson, Samantha Mary.; Chuturgoon, Anil Amichund.; Phulukdaree, Alisa.
Fumonisin B1 (FB1) is a mycotoxin which is well characterised as a contaminant of maize and maize-based products worldwide, especially in South Africa. Its toxic effects have been associated with hepatotoxicity, nephrotoxicity and carcinogenicity. Tissue transglutaminase (TG2) is a unique and ubiquitous enzyme that catalyses the post-translational modification of proteins and has GTPase activity. Tissue transglutaminase is an important enzyme in a number of biological processes such as cell differentiation and proliferation, extracellular matrix organisation, cell signalling and apoptosis. This study investigated the possible role of TG2 induction by FB1 and the effect FB1 toxicity has on the endoplasmic reticulum (ER) stress pathway in HepG2 cells. A SDS-PAGE adaption of the TG2 activity assay confirmed TG2 crosslinking activity by FB1 incorporation into fibronectin in the presence of calcium and TG2. This interaction was validated using fluorescent microscopy where FB1 incorporated into the HepG2 cell’s cytoplasmic vesicles and plasma membrane. The up-regulation of TG2 in HepG2 cells treated with FB1 was further investigated using western blotting and showed increased TG2 up-regulation. Fumonisin B1 disrupts membrane-bound sphingolipids as a mechanism of toxicity; FB1 was shown to cause cytoskeletal damage and disrupted the cell’s membranes leading to cell stress. The protein kinase RNA-like endoplasmic reticulum kinase (PERK) ER stress pathway was induced as a result of FB1 exposure and investigated using western blotting and quantitative polymerase chain reaction. After 72hours with 50μM and 100μM FB1 total PERK decreased, phosphorylated eukaryotic initiating factor α remained activated with a significant increase in messenger RNA (mRNA) expression (p<0.05) and transcription factor CCAAT-enhancer-binding protein homologous protein mRNA was significantly induced (p<0.05). The involvement of nuclear factor kappa B (NFkB) and TG2 in ER stress induced apoptosis was investigated through western blotting and quantitative polymerase chain reaction. After 72hours, an up-regulation of both nuclear NFkB and nuclear TG2 was observed; with a corresponding significant increase in nuclear TG2 mRNA expression (p<0.05). A significant increase in transcription factor, Sp1 mRNA expression (p<0.05) was observed after 72hours. Data suggests PERK activation leads to NFkB induction and nuclear translocation; which promoted nuclear TG2 transcription. The activation of TG2 resulted in Sp1 crosslinks that could act as potential inducers of FB1 induced apoptosis. Flow cytometry was used to measure apoptosis and mitochondrial depolarisation. Caspase activity was measured using the Caspase-Glo® assays and ATP concentration was measured using CellTitre-GloTM assay. After 72hours caspases 3/7 and 8 showed a significant decrease in activity at 100μM FB1 (p<0.05) and a decrease in caspase 9. After 72hours with 10μM FB1 treatment a significant increase in phosphatidylserine externalisation (p<0.05), a significant decrease in healthy/live cells (p<0.05) and a significant increase in depolarised mitochondria (p<0.05) were observed. There was also a significant increase in Sp1 mRNA expression (p<0.05). However, at 50μM FB1 treatment there was a decrease in phosphatidylserine externalisation, a significant increase in live cells (p<0.05) and a significant decrease in depolarised mitochondria (p<0.05). Data suggests that ER stress persisted in HepG2 cells with no apoptosis or cell recovery occurring at high chronic doses of FB1 whilst ER stress induced apoptosis at low chronic doses of FB1 in HepG2 cells. Fumonisin B1 may be a possible substrate for TG2 crosslinking activity due to its primary amine group, since this mycotoxin has the potential to induce TG2 expression and activation. Further studies are required to determine the role of FB1 in the inositol-requiring protein 1α and activating transcription factor 6 arms of the ER stress pathway.
The effects of fumonisin B¹ in preeclampsia.
(2012) Serumula, Metse Regina.; Chuturgoon, Anil Amichund.; Moodley, Jagidesa.
Preeclampsia is the leading cause of foetal and maternal mortality and morbidity in developing countries. In South Africa, maize is a dietary staple for most black African populations and is susceptible to contamination by mycotoxins such as fumonisin B1 (FB1).Fumonisin B1 is a ubiquitous secondary metabolite of Fusarium fungi produced predominantly by Fusarium verticillioides. This mycotoxin shares structural similarities with the backbone of sphingoid bases (sphinganine and sphingosine) which are substrates for the biosynthesis of complex sphingolipids. The mechanism of FB1 toxicity therefore is centred on the disruption of this process. The aim of the present study was to elucidate the possible causal link between FB1 and preeclampsia. Following ethical approval, 20 normotensive and 20 preeclamptic patients were recruited into the study. Blood and placental tissue were collected and processed for further analysis. The presence of FB1 was verified using standard immunohistochemical and electrophoretic techniques. The levels of FB1 and sphingolipids were quantified using high performance liquid chromatography (HPLC). Western blotting was conducted to confirm the presence of FB1 in the serum. Placental tissue apoptosis was evaluated using Hoechst staining and other markers. Lipid peroxidation was measured in serum and placental tissue of both groups. Fumonisin B1 was immunolocalised within the endothelial cells and mesenchymal cells of placentas from both groups, while FB1 was present in cytotrophoblastic cells of preeclamptic patients only. In addition, FB1 concentrations were significantly higher in preeclamptic compared to normotensive serum samples. Sphinganine was significantly elevated in preeclamptic serum samples whilst there was no statistical difference in the sphingosine levels between the groups. Chromatin condensation was higher in the preeclamptic patients. Caspase 3 and Fas were present with greater intensity in preeclamptic samples. The levels of lipid peroxidation were significantly higher in both serum and placental tissue of preeclamptic patients. This study has demonstrated not only the presence of FB1 in the serum and placental tissues of pregnant women but also the potential effects of this mycotoxin in the humans.
The effects of Sutherlandia frutescens in cultured renal proximal and distal tubule epithelial cells.
(2009) Phulukdaree, Alisa.; Chuturgoon, Anil Amichund.
Sutherlandia frutescens (SF), an indigenous medicinal plant to South Africa (SA), is traditionally used to treat a diverse range of illnesses including cancer and viral infections. The biologically active compounds of SF are polar, thus renal elimination increases susceptibility to toxicity. This study investigated the antioxidant potential, lipid peroxidation, mitochondrial membrane potential and apoptotic induction by SF on proximal and distal tubule epithelial cells. Cell viability was determined using the MTT assay. Mitochondrial membrane potential was determined using a flow cytometric JC-1 Mitoscreen assay. Cellular glutathione and apoptosis were measured using the GSH-GloTM Glutathione assay and Caspase-Glo® 3/7 assay, respectively. The IC50 values from the cell viability results for LLC-PK1 and MDBK was 15 mg/ml and 7 mg/ml, respectively. SF significantly decreased intracellular GSH in LLC-PK1 (p < 0.0001) and MDBK (p < 0.0001) cells. Lipid peroxidation increased in LLC-PK1 (p < 0.0001) and MDBK (p < 0.0001) cells. JC-1 analysis showed that SF promoted mitochondrial membrane depolarization in both LLC-PK1 and MDBK cells up to 80% (p < 0.0001). The activity of caspase 3/7 increased both LLC-PK1 (11.9-fold; p < 0.0001) and MDBK (2.2-fold; p < 0.0001) cells. SF at high concentrations plays a role in increased oxidative stress, altered mitochondrial membrane integrity and promoting apoptosis in renal tubule epithelia.
The effects of Tulbaghia violacea (wild garlic) leaf and bulb extracts on an oesophageal cancer cell line (SNO)
(2012) Moonsamy, Suri.; Myburg, Rene Bernadette.; Serumula, Metse Regina.
Ethnopharmacological relevance: Indigenous plants such as Tulbaghia violacea(TV) and Allium sativum (garlic) are traditionally used as natural remedies to treat a variety of ailments, including cancer. This study investigated the effects of TV leaf and bulb extracts and garlic extract on a cancerous oesophageal cell line (SNO). Materials and methods: The methylthiazoltetrazolium (MTT) assay was used to determine the IC50 of TV leaf (TVL) (250μg/ml) and TV bulb extracts (TVB) (25μg/ml) and garlic (500μg/ml). Extracts were treated individually and in combination for a period of 24 hours. Oxidative damage and intracellular glutathione levels were assessed using the Thiobarbituric Acid Reactive Substances (TBARS) Assay and GSH-Glo™ Luminometry Assay, respectively. The CellTiter-Glo® Luminescent Cell Viability Assay was used to assess ATP activity. Induction of apoptosis and mitochondrial membrane potential were determined via the Caspase-Glo® 3/7 Assay, Caspase-Glo® 8 Assay, Caspase-Glo® 9 Assay and JC-1 Mitoscreen Assay, respectively. Morphological apoptotic changes were determined using the Hoechst 33342 stain. Expressions of p53, PARP and NFKB activities were determined by western blotting. Results: Bulb and leaf extracts of TV increased lipid peroxidation compared to the control (p>0.05), whilst garlic and combination of TV leaf and bulb (TVB + TVL) extracts significantly decreased lipid peroxidation relative to the control (p< 0.05). Endogenous glutathione levels significantly decreased in all TV treatments compared to the control (p<0.05).However, garlic was accompanied by insignificantly increased intracellular glutathione levels compared to the control (p> 0.05). The percentages of depolarised mitochondria in all treated cells were significantly decreased compared to untreated cells (p< 0.05). ATP levels increased significantly in garlic and combination (TVB + TVL) treated cells as compared to the control (p< 0.05), yet no significant differences were noted in TVL and TVB treatments (p> 0.05). Caspase8 and caspase 9 activities significantly increased in garlic and combination treated cells relative to the control (p<0.05). A similar trend was noted for caspase 3/7 activity in garlic and combination treatments (p< 0.05). However, initiator and executioner activities in TVL (p> 0.05) and TVB (p> 0.05) treatments did not significantly differ from the control (p> 0.05). All treatments (including garlic) resulted in increased DNA fragmentation and condensation. All treatments decreased p53 expression (p< 0.05), PARP expression (p< 0.05) and NFK B expression (p>0.05) compared to the control. Conclusions: All TV extracts and garlic induces apoptosis in the oesophageal cancerous SNO cell line through changes in oxidative stress, antioxidant systems, and nuclear chromatin condensation, as well as through induction of nuclear genes and signalling pathways. Since inhibition of apoptosis is a principal alteration in cancer, induction of apoptosis would result in a decrease in cancer cell growth. Thus, TV could be exploited as a potential anti-cancer agent.
The effects of Tulbaghia violacea leaf, bulb and stalk extracts on Jurkat cells.
(2012) Mackenzie, Jared Stuart.; Myburg, Rene Bernadette.; Serumula, Metse Regina.
Studies have shown that the traditional healers have used Tulbaghia violacea (TV) (also known as ‘wild garlic’) for the treatment of a number of ailments including fever, tuberculosis, stomach problems, and oesophageal cancer. However, little is known with regards to the anticancer and antiproliferative properties of this plant. Therefore, this study investigated the effects of TV and domesticated garlic extracts on Jurkat cells, in order to determine whether or not these extracts possess anti-proliferative properties. Cultured Jurkat cells were treated with IC50 concentrations of garlic (14μg/ml), TV leaf (256μg/ml), TV bulb (225μg/ml) and TV stalk (216μg/ml) extracts as determined by the methylthiazol tetrazolium assay. Free radical production was measured using the thiobarbituric acid reactive substance (TBARS) and nitric oxide (NO) assays, while glutathione (GSH) concentration was measured using the GSH-Glo™ assay. The apoptosis inducing properties of each extract were measured using flow cytometry (Annexin V- Fluos and JC-1 assays) and luminometry (caspases 3/7, 8, 9 and ATP). Western blots were run to determine protein expression, while comet and DNA fragmentation assays were used to determine the level of DNA damage induced. Wild and domesticated garlic extracts induced a significant increase in malondialdehyde concentration ([MDA]), with TV bulb extract inducing the highest concentration (p<0.0001). A significant increase in NO concentration was observed in the bulb (p<0.0001) and stalk (p<0.001) extracts, and leaf (p<0.05) and stalk (p<0.05) TV extracts significantly increasing GSH concentration. The longest comet tails were observed in TV bulb extracts (p<0.0001) and comprised mainly of single strand breaks, while the comets induced following garlic exposure contained double strand breaks. All extracts, except TV leaf, increased the percentage of cells undergoing apoptosis. Tulbaghia violacea leaf induced a significant (p<0.0001) increase in percentage of cells undergoing necrosis, whereas TV bulb resulted in a significant (p<0.0001) decrease. All TV extracts induced caspase 3/7 and 9 activity, with the most significant increase in caspase 9 activity observed for TV leaf and bulb. No significant change in caspase 3/7 activity was evident for domesticated garlic. Cleavage of PARP and expression of NF B and HSP 70 occured for all extracts. However, HSP 70 was not differentially expressed. Exposure to wild and domesticated garlic extracts induced peroxidative lipid and DNA damage within the cells, indicating oxidative stress. This damage occurred in conjunction with increased percentage of cells undergoing apoptosis and expression of caspase 3/7. Therefore, these findings suggest that TV is inducing cell death through apoptosis in Jurkat cells using a number of mechanisms, including the induction of oxidative stress. This is of clinical significance, as cell death through apoptosis is the preferred method of action for anti-cancer drugs.
Exploring Iso-mukaadial acetates and other small compounds as inhibitors of recombinant Plasmodium falciparum lactate dehydrogenase.
(2021) Mabaso, Nonduduzo Hlengiwe.; Mhlongo, Ndumiso Nhlakanipho .; Pooe, Ofentse Jacob.
Malaria is a major killer disease in Sub-Saharan Africa, this disease is caused by a protozoan parasite of genus Plasmodium. It is a pressing health issue the public is facing, and the effectiveness of every treatment developed thus far is being jeopardized by the emergence of parasite drug resistance. This then creates a demand for new antiprotozoal medication, necessitating novel approaches that will assure the long- term discovery of the lead compounds. The investigation of compounds such as Iso-mukaadial acetate (IMA), Betulinic acid (BA), Ursolic acid (UA) and Oleanolic acid (OEA) which are isolated from plants shows to possess antimalarial activity. These compounds either originate from various plants or leaves, IMA which is isolated from a pepper bark tree, BA from bark of a plant species (white birch), UA from leaves of (lavender, rosemary), and OEA found in leaves and Olea europaea fruit. This study aims to investigate the inhibitory properties of these compounds against Plasmodium falciparum lactate dehydrogenase (PFLDH) an enzyme found in the parasite glycolytic pathway that converts pyruvate to lactate and in so doing, provides the energy needed for the survival of the malarial parasite. These methodologies were followed to conduct this study; Recombinant PfLDH was expressed and then purified for further analysis including colony PCR, expression, purification, interaction studies including Fourier transform infrared (FTIR) analysis and Ultraviolet-visible spectroscopy (UV-Vis), antimicrobial activity along with in silico analysis. The following results were obtained: Colony PCR confirmed the presence of a 951bp insert in the PKK223 plasmid. Metal affinity chromatography successfully purified PfLDH protein sized 34.9kDa which was confirmed by ExPasy ProtParam server. The following results were obtained from isolated compounds (BA and IMA) that were screened for IC50 to demonstrate overall activity against the asexual P. falciparum. BA and IMA had IC50 values of 1.27 and 1.03μg/ml against asexual P. falciparum, respectively. When compounds were incubated with protein, FTIR analysis showed a clear shift in the curve, which is indicative of an interaction between IMA and BA with PfLDH. UV-Vis showed that structural conformational change was induced, resulting in an interaction of the compounds with the aromatic side chains of PfLDH. The in silico analysis showed where these interactions occurred, highlighting the ligand atoms responsible for the interaction. Based on these findings, it is possible that these investigated compounds could be effective PfLDH inhibitors as they have binding affinities which are like the standard drug, chloroquine (QA).
Fumonisin B1 induced antioxidant response in C57BL/6 male mice brain.
(2018) Sibiya, Thabani.; Chuturgoon, Anil Amichund.; Nagiah, Savania.; Ghazi, Terisha.
Background: Fumonisin B1 (FB1), a mycotoxin produced by the Fusarium species, contaminates maize. In South Africa maize is a dietary staple and FB1 endangers human and animal health. FB1 is known to have neurodegenerative effects; inhibits mitochondrial respiration, causes mitochondrial membrane depolarization and excessive ROS production. This study investigated the antioxidant response in mice brain after acute (24 hrs) and prolonged (10 days) exposure to FB1. Methods: Four groups (Control acute, FB1 acute, Control prolonged, FB1 prolonged) of C57BL/6 male mice (n=5 per group) were used. All controls were orally administered 0.1M PBS and FB1 groups were administered 5mg/kg of FB1. Following acute and prolonged exposure, the mice were euthanised by halothane anaesthesia. Brain tissues were harvested and stored in Qiazol and Cytobuster for RNA and protein isolation, respectively. Protein expression of CAT, pNrf2 and Nrf2 were determined using western blots. The mRNA expression of Nrf2, miR-141, SOD2, GPx, Tfam, LON, SIRT3 and Tau wwere determined using qPCR. Results: Protein expression of Nrf2 (Acute: *p=0,0144; prolonged: **p=0,0094) and pNrf2 (acute: *p=0,0132; prolonged: *p=0,0462) was significantly increased upon 24 hrs and significantly decreased upon 10 days in tissue exposed to FB1, while mRNA levels of Nrf2 were significantly reduced upon acute (***p=0,0001) and prolonged (**p=0,0013) exposure. FB1 induced a significant decrease in miR-141 levels in tissue following acute (**p=0,0019) and prolonged (***p=0,0004) exposure. FB1 increased the protein expression of CAT in tissue following acute (p=0,1206) and significantly increased expression upon prolonged (**p=0,0010) exposure. FB1 also significantly increased the mRNA expression of GPx in acute (***p=0,0001) and prolonged (**p=0,0024) exposure. FB1 significantly decreased the expression of SOD2 in mice brain following acute (**p=0,0070) and non-significantly decrease upon prolonged (p=0,2725) exposure. Tfam and LONP1 levels were significantly decreased upon acute (***p=0,0003, ***p=0,0005) and prolonged (*p=0,0196, *p=0,0117) exposure to FB1 respectively. However, SIRT3 expression was decreased upon acute (p=0,0594) and significantly increased upon prolonged (*p=0,0283) exposure to FB1.The mRNA expression of tau was significantly reduced upon acute (**p=0,0054) and prolonged (*p=0,0273) exposure to FB1. Conclusion: FB1 compromises antioxidant and mitochondrial survival responses in mice brain. This may have implications in FB1-induced neurodegeneration.
Fumonisin B1-induced oxidative stress in human liver (HepG2) cells – an alternate mechanism of carcinogenesis.
(2017) Arumugam, Thilona.; Chuturgoon, Anil Amichund.; Nagiah, Savania.
Abstract available in pdf.
Fumonisin B2 induces mitochondrial stress and mitophagy in Hek293 cells.
(2019) Mohan, Jivanka.; Chuturgoon, Anil Amichund.; Sheik-Abdul, Naeem.
Food insecurity poses a significant socio-economic problem in third world economies, particularly in countries that rely heavily on maize and maize products. Ubiquitous soil fungi parasitize agricultural commodities and produce mycotoxins. Fumonisin B2 (FB2), a neglected mycotoxin, is produced by several Fusarium species. The aim of this study was to investigate mitochondrial stress responses in human embryonic kidney (Hek293) cells exposed to FB2 for 24 hours (hr). Cell viability was assessed via the methylthiazol tetrazolium (MTT) assay and the half maximal inhibitory concentration (IC50) value (317.4 μM) was generated. Additional concentrations of 100 μM and 500 μM were selected to achieve a broader toxic profile of FB2. Reactive oxygen species (ROS) was quantified (fluorescence), mitochondrial membrane depolarisation (fluorescence) was assessed and adenosine triphosphate (ATP) concentration was evaluated (luminometry) to assess mitochondrial integrity. The relative expression of mitochondrial stress response proteins, Sirtuin 3 (SIRT3), Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), LON protease (LONP1), PTEN-induced putative kinase 1 (PINK1), ubiquitin-binding adaptor p62 (p62) and heat shock protein 60 (HSP60) was determined by western blots. Transcript levels of SIRT3, PINK1 and microRNA-27b (miR-27b) was assessed using quantitative PCR (qPCR). Results indicated that both low and high concentrations of FB2 that were within the naturally occurring concentration range of the compound were able to induce mitochondrial dysfunction. FB2 (IC50) downregulated mitochondrial stress proteins and upregulated mitophagy markers. Despite upregulation of mitochondrial stress maintenance proteins at the highest concentration (500 μM) of FB2, mitophagic markers increased with subsequent cell death; whilst at a lower concentration (100 μM) of FB2, mitochondrial stress protein expressions were upregulated resulting in decreased expression of mitophagic markers and cell proliferation. In conclusion, FB2 was cytotoxic to the kidney derived Hek293 cells via induction of mitochondrial stress and mitophagy. Keywords: Fumonisin B2, mitophagy, mitochondrial stress, PINK1, Nrf2, SIRT3, human kidney cells, microRNA
Fusaric acid dampens the Nrf2-mediated stress response in human embryonic kidney cells.
(2016) Govender, Melissa.; Chuturgoon, Anil Amichund.; Nagiah, Savania.
Abstract available in PDF file.
Fusaric acid Fumonisin B1 CO -treatment regulates AMPK signalling and induces Apoptosis in HEPG2 cells.
(2019) Shilabye, Patane Sylvester.; Chuturgoon, Anil Amichund.; Ghazi, Terisha.
Background/Aim: Fusaric acid (FA) and Fumonisin B1 (FB1) are mycotoxins produced by Fusarium fungal species. These mycotoxins are major contaminants of maize and contribute to toxicity in animals and humans. The main mechanisms of FA and FB1 toxicity involve the induction of oxidative stress and apoptosis; however, FA was additionally found to chelate divalent cations, whereas FB1 inhibits sphingolipid synthesis. AMPK is an energy sensor involved in regulating cell proliferation. AMPK targets the transcription factors, p53 and FOXO3a that play a major role in apoptosis. To date numerous studies have investigated the individual effects of FA and FB1, however, their combined synergistic effects are unclear. This study investigated the effect of FA and FB1 co-treatment on AMPK-induced apoptosis in liver HepG2 cells. Methods: HepG2 cells were cultured and co-treated with various concentrations (5, 27, 100μM and combined 104μM FA and 200μM FB1 IC50s) of FA and FB1 for 24 hrs. Cytotoxic effects of FA and FB1 on HepG2 cells were determined using the MTT assay. The TBARS assay was used to determine oxidative stress. Western blot was used to determine protein expression of AMPK, p-AMPK and p53, whereas q-PCR was used to measure FOXO3a mRNA expression. LDH assay was used to measure membrane integrity. ATP levels and activity of caspases -3/7, -8 and -9 were measured using luminometry. Results: A combination of FA and FB1 decreased cell viability in a dose dependant manner. An IC50 of 27μM for FA and FB1 was obtained. ATP levels were significantly increased at 5μM and 27μM, whereas at 100μM and combined IC50s were significantly decreased (p<0.0001). Oxidative stress was significantly increased in FA and FB1 treated cells in a dose dependent manner (p<0.0001). The protein expression of total AMPK was decreased at 5μM, but increased at 27μM, 100μM and combined IC50s in relation to control (p<0.0001).p- AMPK showed a significant decrease (p<0.0001) in all FA and FB1 treated samples despite the increase in the expression of total AMPK. FOXO3a mRNA expression was decreased at 5μM and at combined IC50s, with the decrease being significant at 5μM. The results also indicated an increase at 27μM and 100μM (p<0.0001). p53 protein expressions were significantly decreased in all samples (p<0.0001). Caspase -3/7, -8 and -9 were significantly increased at 5-100μM and decreased at combined IC50s in HepG2 cells. In FA and FB1 samples, LDH levels were significantly decreased at 5μM and 27μM, and significantly increased at 100μM and combined IC50s (p<0.0001). Conclusion: FA and FB1 co-treatments suppressed AMPK signalling by downregulating p- AMPK and induced apoptosis and/necrosis in HepG2 cells.

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