Masters Degrees (Medical Biochemistry)

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The effect of patulin on oxidative stress and global DNA methylation in C57BL/6 mice hearts.
(2024) Radebe, Noxolo Laurah.; Ghazi, Terisha.; Chuturgoon, Anil Amichund.
Mycotoxins are chemical compounds produced by fungi that, when ingested, induce a variety of diseases including hepatotoxicity, nephrotoxicity, gastrointestinal toxicity, neurotoxicity, and immunotoxicity. Patulin (PAT) is a common mycotoxin produced by Penicillium, Aspergillus, and Byssochlamys fungal species, and is commonly found in apples and its products. Researchers have reported that consumption of PAT-contaminated foods causes nephrotoxicity and hepatotoxicity; however, its effects on the heart are unknown. Following reports that PAT causes oxidative stress and epigenetic changes, this study assessed if PAT induces oxidative stress and global DNA methylation in C57BL/6 mice hearts. Methods included: (i) ten mice treatment with PAT (2,5mg/kg) and PBS (0,1M) for 24hr, five mice per treatment group, (ii) thiobarbturic acid reactive substances (TBARS) assay (lipid peroxidation), (iii) quantitative polymerase chain reaction (gene expression of kelch-like ECH-associated protein 1 (Keap1), nuclear factor erythroid 2-related factor 2 (Nrf2), glutathione peroxidase (GPx), superoxide dismutase 2 (SOD2), catalase (CAT), methyl-CpG binding domain protein 2 (MBD2), DNA methyltransferases (DNMT1, DNMT3A, DNMT3B), (iv) western blot (protein expression of Nrf2, SOD2, CAT, MBD2, DNMT3A and DNMT1), (v) enzyme-linked immunosorbent assay (global DNA methylation levels). PAT caused an induction of oxidative stress as indicated by the significant increase in malondialdehyde (MDA) levels (0.52-fold, p = 0.005). PAT significantly increased gene expression of Nrf2 (1.20-fold, p=0.049), GPx (1.20-fold, p = 0.028), SOD2 (1.40-fold, p = 0.042) and CAT (0.90-fold, p = 0.038); and significantly decreased gene expression of keap1 (1.10-fold, p = 0.007). PAT increased expression of Nrf2 protein, significantly (0.64-fold, p = 0.005), SOD2 (2.40-fold, p = 0.020), and CAT (1.88-fold, p = 0.004). Additionally, PAT suppressed the gene expression of DNMT3A (0.41-fold, p = 0,0001) and MBD2 (0.45-fold, p = 0,0001) and increased the gene expression of DNMT1 (1.48-fold, p = 0,02) and DNMT3B (1.62-fold, p = 0,04). PAT increased the protein expression of DNMT1 (0.68-fold, p=0.029); and significantly decreased the protein expression of MBD2 (0.48-fold, p=0.008) and DNMT3A (0.50-fold, p = 0.0001). The alterations in DNMTs and MBD2 led to PAT-induced global DNA hypermethylation (1.72-fold, p = 0,02). In conclusion, PAT induced oxidative stress and global DNA hypermethylation in C57BL/6 mice hearts and is toxic to the heart.
Artemisia Afra crude aqueous extract induces NRF2-mediated antioxidant defence against oxidative stress and inhibits apoptosis via upregulation of hsp27 and HSP90 in A549 lung cancer cells.
(2024) Nkambule, Awande Comfort Monwabisi.; Khan , Rene.; Kumalo, Hezekiel Mathambo.
Lung cancer ranks first in mortality rates across all cancer types globally. Currently available treatment options are associated with undesirable outcomes for patients such as treatment related adverse events, low efficacy, and high costs. There have been ongoing efforts in novel drug discovery and development research using medicinal plants as a source for anti-tumour agents that will improve on these aspects. One such promising lead in this space is the South African plant Artemisia afra. In this study, the underlying mechanisms associated with the cytotoxicity of Artemisia. afra in A549 lung cancer cells were investigated following 48 hours exposure to the plant’s crude aqueous extract. Cytotoxicity was assessed using the MTT, ATP and mitochondrial membrane potential (ΔΨm) assays, and CYP3A4 activity was ascertained by luminometry. The free radical production and antioxidant response was determined using the TBARS, nitrates, luminometry and western blotting assays to measure ROS, RNS, GSH and antioxidant protein expression (SOD2, Gpx1, catalase), respectively. The Nrf2, HSP27 and HSP90 protein expression was detected by qPCR or western blotting. Cell death parameters such as caspase activity, phosphatidylserine (apoptosis) and necrosis were quantified by luminometry, and Bcl2 family proteins were ascertained by western blotting. The IC50 as per the dose-dependent MTT curve was determined to be at 260 μg/ml. Increased Artemisia afra metabolism by CYP3A4 (p<0.05) was observed in treated cells compared to the control, but ATP production was decreased (p<0.05) despite minimal changes in the ΔΨm. The Artemisia afra treatment significantly increased ROS production (p<0.05), which was met with a reactive spike in Nrf2 (p<0.05) gene expression. Concurrent increase in SOD2, Gpx1 (p<0.05) and catalase (p<0.05) protein expression, as well as GSH were noted. Little difference was recorded in the levels of nitrates. Pro-apoptotic Bax (p<0.05) was upregulated, while anti-apoptotic Bcl2 (p<0.05) was reduced. Intriguingly the initiator caspases, caspase 8 (p>0.05) and caspase 9 (p<0.05) were decreased in treated cells, in accordance with increased potent anti-apoptotic proteins HSP27 (p<0.05) and HSP90 (p<0.05), positing their role in inhibiting the initiator caspases but xIAP was not changed. Apoptosis was excluded as the major form of cytotoxicity in the treated cells given that the results showed a reduction in execution caspases, caspases 3/7 (p<0.05) and reduced PS externalisation (p<0.05). Necrosis was also excluded as the mode of cytotoxicity as necrosis, even though LDH levels were increased in the Artemisia afra treated A549 cells. The MTT and ATP assay analysis revealed a decline in cell viability after 48 hours of treatment. An antioxidant response was observed, but ROS were increased. However, cell death by apoptosis was prevented by HSP. Taken together the current study confirmed that the Artemisa afra crude aqueous extract mediated cytotoxicity by oxidative stress, but demonstrated anti-apoptotic effects via HSP in A549 cells.
The effect of patulin on adrenergic receptor signalling and DNA methylation in C57BL/6 mouse livers.
(2024) Naidoo, Alisha.; Chuturgoon, Anil Amichund.; Ghazi, Terisha.
Background: Secondary metabolic products of fungi and mould are called mycotoxins, and they are usually hazardous to organisms. Patulin (PAT) is a mycotoxin most prevalent in apples and their products, such as juice and cider. PAT may threaten animal and human health by causing chronic effects including immunotoxicity, genotoxicity, teratogenicity, and carcinogenicity. Research demonstrates that PAT is hepatotoxic; however, its mechanism of action is unclear. The adrenergic receptors are altered during liver injury. The adrenergic receptors utilise the mitogen- activated protein kinases/extracellular signal-regulated kinase (MAPK/ERK) and phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) pathways. It is unclear if ARs are modulated by DNA methylation. DNA methylation is crucial for cell differentiation. Hence its dysregulation can lead to diseases such as cancer. Aim: To determine the effect of PAT on global DNA methylation and adrenergic receptor signalling in C57BL/6 mouse livers. Methods: In the livers of four C57BL/6 mice fed PAT (2.5 mg/kg BW) for 10 days: qPCR determined the mRNA expression of alpha and beta-adrenergic receptors, as well as MAPK, MAPK14, ERK1/2, PI3K and AKT, DNMT1, DNMT3A, DNMT3B and MBD2. Western blot determined the protein expression of P38, ERK1/2, PI3K, DNMT1, DNMT3A, and MBD2. The ELISA assay was used to determine global DNA methylation levels. Results: PAT significantly increased alpha-1A adrenergic receptor mRNA levels whilst decreasing alpha-2A, 2B, and all beta-adrenergic receptor expression, with beta-2 reduced significantly. PI3K’s decline was PAT-induced. PAT significantly increased AKT, MAPK, MAPK14, and ERK1 expression but significantly reduced ERK2 levels. PAT increased MBD2 and DNMT1 expression while significantly decreasing DNMT3A and 3B levels. PAT significantly increased and decreased the protein levels of P38 and ERK1/2 respectively. Additionally, a PAT- mediated increase in PI3K protein levels was observed. PAT significantly increased DNMT1 and increased DNMT3A and MBD2 expression. Significant global hypermethylation was PAT- induced. Discussion/Conclusion: PAT significantly impacted alpha-1A and beta-2 adrenergic receptors which also utilise the MAPK/ERK/PI3K/AKT pathway. The dysregulation of these signalling cascades has been associated with altered expression of the adrenergic receptors. PAT-induced global hypermethylation. PAT disrupts adrenergic receptor signalling and modifies global DNA methylation, thus resulting in liver injury.
Monsonia burkeana induces caspase-dependent apoptosis in Caco- 2 cells and nitrosative stress-induced necroptosis in HepG2 cells.
(2023) Naicker, Mayanka.; Khan, Rene.; Kumalo, Hezekiel Mathambo.
Introduction: Cancer is one of the leading causes of death globally. Increased incidence and mortality rates of colorectal and liver cancer have been reported in South Africa and worldwide. Cytotoxic side effects associated with current treatments have sparked interest in using plant phytochemicals as a potential alternate, cost-effective cancer therapeutic. Monsonia burkeana Planch. Ex Harv, also known as "special tea", is a medicinal plant native to southern Africa that treats various ailments. It has also shown potential application in anticancer therapy. This study investigated the anticancer effects of M. burkeana crude aqueous extract in the Caco-2 and HepG2 cell lines. Methods: The target cells were reconstituted in CCM and treated with 0 - 5000μg/ml of M.burkeana plant extract for 48 hours during the MTT assay to obtain the 20% and 50% inhibitory concentration (IC20 and IC50), which was used for experiments that followed. The cytotoxic effects were further evaluated using the LDH and CYP3A4 activity assay to determine oxidative breakdown and assess the ATP and JC-10 levels to measure mitochondrial integrity. The antioxidant response of M. burkeana involved conducting the TBARS/NOS assay to extrapolate reactive oxygen and nitrogen species, GSH quantification, and western blotting to detect (SOD, NRF2, iNOS) protein expression. The mRNA gene expression of Gpx and OGG1 was evaluated using qPCR. To assess cell death, the Annexin-V assay distinguished apoptotic and necrotic cells, caspase activation assays were conducted as a marker of apoptosis, and western blotting determined the expression of the following proteins: p53, p-p53, BAX, NFB, cIAP2, cleaved caspase 3 and BCL-2. Gene expression of MLKL, RIP1, RIP3, NFB, and TNF-α was also assessed to evaluate its role in cell death. Lastly, data analysis was conducted using statistical tests in GraphPad Prism. Results: Cytochrome P450 3A4 activity increased for both cell lines, causing a dose-dependent decrease in cell viability in Caco-2 cells and HepG2 cells, with an IC50 value of 293.8 μg/ml and an IC20 value of 169.8 μg/ml in Caco-2 cells. In HepG2 cells, the IC50 value was 335.4 μg/ml of M. burkeana extract, and the IC20 value was 154.9 μg/ml. The decreased ATP concentration in Caco-2 and HepG2 cells for both treatments was consistent with the increased ΔΨm, confirming a reduced metabolic activity. Decreased MDA levels indicating lipid peroxidation occurred for both cell lines, while increased OGG1 for the Caco-2 IC20 treatment suggests DNA oxidation for this treatment only. Nitrite levels decreased for both treatments in Caco-2 cells but increased in HepG2 cells. There was also a decrease in iNOS protein expression for both cell lines. Membrane disruption was validated by increased LDH for both cell lines and HepG2 cells were associated with RNS-induced membrane disruption. Oxidative stress is implied due to decreased GSH concentration and upregulation of SOD2 protein expression at both treatment concentrations for Caco-2 and HepG2 cells. However, Gpx-1 decreased for Caco-2 cells and the IC20 treatment in HepG2 cells. There was an associated upregulation of NRF-2 protein expression for both cell lines at the IC50 treatment concentrations to regulate antioxidant proteins. Initiator caspase 8 activity increased for both treatment concentrations in Caco-2 cells, implying that in Caco-2 cells, apoptosis was stimulated via the extrinsic pathway. In addition, intrinsic apoptosis was initiated in the IC20-treated Caco-2 cells as caspase 9 activity increased. Caspase 8 and caspase 9 activity decreased for both treatment concentrations in HepG2 cells. The p-p53/p53 ratio decreased for both treatments in Caco-2 cells. Thus, p53 did not mediate the transcription of pro-apoptotic BCL-2 family genes such as BAX, which dropped in both treatment concentrations. Anti-apoptotic BCL-2 was also reduced in Caco-2 cells. In HepG2 cells, the protein expression of p-p53/p53 remained relatively unchanged for IC20- treated cells and increased for IC50-treated, but the BAX decreased for IC20, and BCL-2 protein expression increased for the IC50 treatment concentration. M. burkeana facilitated the execution of apoptosis in both cell lines, as caspase 3/7 was increased and phosphatidylserine was externalised, but necrosis was also increased. There was downregulation of TNF in Caco-2 cells, and decreased RIPK1, RIPK3 and MLKL for both treatment concentrations corresponding with increased cIAP2. In HepG2 cells, there was an increase in RIPK1 gene expression for both the IC20 and IC50 treatment concentrations, decreased cIAP2 protein expression, increased RIPK3 and MLKL gene expression. Conclusion: According to the results, M. burkeana crude aqueous extract caused caspase-dependent apoptosis in Caco-2 cells and nitrosative stress-induced necroptosis in HepG2 cells, which validates that M. burkeana can be further explored as an anticancer therapeutic.
Cyclen tetrahydrochloride induces necroptosis via oxidative and nitrosative stress in mcf-7 and MDA-MB breast cancer cells respectively.
(2023) Munsamy , Mikayla Libby.; Khan, Rene.; Kumalo, Hezekiel Mathambo.
Introduction: Breast cancer (BC) is a heterogeneous disease categorised based on the availability of specific female hormone receptors and is thus predominantly associated with female mortality and morbidity globally. The progression of BC and conventional treatments are subtype-specific, producing immunocompromising effects often linked to oxidative stress and cell death pathways, including necroptosis. A promising therapeutic synthetic compound 1,4,7,10-tetraazacyclododecane (cyclen) tetrahydrochloride is a divalent metal chelator that exhibits potential multi-target anti-cancer activities. It harbours high binding affinities toward transition-metal ions or cations, an advantageous pathway to be explored in biomedical research. Aim: This study aims to determine the anti-proliferative and antioxidant mechanisms of cyclen tetrahydrochloride in MCF-7 and MDA-MB-231 human breast cancer cells. Methods: The MTT assay assessed the cell viability of MCF-7 and MDA-MB-231 cells following exposure to cyclen tetrahydrochloride (0-1000μM) for 48 hours. Luminometric analysis of ATP and Δm ascertained mitochondrial integrity. Cells were assayed for free radical production (TBARS and NOS assays), while reactive nitrogen species (RNS) were verified by western blotting for iNOS. The antioxidant response was evaluated luminometrically (GSH) and by western blotting (Nrf2 and SOD2). In addition, signalling and cell death pathways activated by oxidative stress (caspases and externalised phosphatidylserine) were evaluated. Cell death by necroptosis was validated by qPCR analysis of RIPK1, RIPK3, MLKL, TNF-α, NF-κB, Gpx-1 and OGG1 gene expression. Results: Cell viability decreased with increasing doses of cyclen tetrahydrochloride treatments for MCF-7 (IC50 = 168.4μM, IC20 = 41.69μM) and MDA-MB-231 (IC50 = 561μM, IC20 = 302.9μM) cells. This was associated with non-significant changes in CYP34A activity and a dissipated Δm in MCF-7 cells. Although Δm was similar to the control in MDA cells, a corresponding decrease in ATP production was noted for both cell lines (p<0.05). Significant increases in MDA concentration (p<0.05) suggested lipid peroxidation associated with ROS production, particularly in MCF-7 cells. Further evidence of increased ROS was implied by increased OGG1 gene expression and decreased GSH, suggesting that oxidative stress was induced. Nitrosative stress was not evident in MCF-7 since iNOS was downregulated, and RNS were decreased. However, cyclen tetrahydrochloride upregulated iNOS to facilitate RNS production in MDA-MB-231 cells (p<0.05) and was associated with increased NF-κB gene expression (p<0.05). An inadequate antioxidant defence was demonstrated by decreased SOD2 and Nrf2 in both cell lines and Gpx-1 gene expression was upregulated in MCF-7 cells only (p<0.05). The prevailing oxidative stress did not initiate apoptosis; caspase-8 and -9 activity were decreased in both cell lines. Although caspase 3/7 was decreased for IC20 MCF-7 cells, the IC50 induced caspase 3/7 and apoptosis was executed owing to increased PS externalisation. In MDA-MB-231 cells, caspase 3/7 increased in the IC20 only, and phosphatidylserine levels were similar to the control. Interestingly, evidence of necrotic cell death in both cell lines was presented by increased DNA fluorescence and LDH leakage (p<0.05). Thus, necroptosis was investigated as an alternate mode of cell death. Significant increases in gene expression of TNF-α (p<0.05), RIPK1 (p<0.05), RIPK3 (p<0.05), and MLKL (p<0.05) demonstrated that necroptosis was increased. Conclusion: Cyclen tetrahydrochloride induced ROS-mediated necroptosis in MCF-7 cells, but ROS and RNS facilitated necroptosis in MDA-MB-231 cells.
Fusaric acid alters global N6- methyladenosine RNA methylation and PI3K/AKT signalling in U87MG cells.
(2024) Matadin, Mcaylin.; Ghazi, Terisha.
Mycotoxins are a global concern due to the extensive damage and loss they cause in the agricultural sector. Mycotoxins are transferred from animal feed into animal-derived consumables,such as eggs, milk and meat presenting a danger to humans. The picolinic acid derivative Fusaric acid (FA), produced by various species of Fusarium, poses a significant risk to both animal and human health because of its toxicological effects on various tissues. It is noted for its unusually potent phytotoxicity in plants and shows prevalence in causing hepatotoxicity, genotoxicity and nephrotoxicity in humans. However, the effects on arguably the most important organ in the human body, the brain, remains incompletely understood. The study set out to investigate the cytotoxic effects of FA on U87MG human glioblastoma cells by monitoring alterations in global m6A RNA methylation as well as gene and/or protein expression levels of the m6A complex and the PI3K/Akt pathway. Methods comprised of (i) culture of U87MG cells; (ii) MTT assay (IC50: 180 μg/ml FA, 24 hrs) which was then used for subsequent treatments; (iii) ELISA; (iv) qRT-PCR (quantify mRNA expression of METTL3, METTL14, YTHDF1, YTHDF2, YTHDF3, FTO, WTAP, YTHDC1, YTHDC2, ALKBH5, BDNF and CREB); (v) western blot (protein expression of BDNF, P-AKT, PCREB and PI3K). FA caused an upregulation (2.1059-fold; p = 0.0150) of global m6A RNA methylation in U87MG cells relative to the control. FA caused a downregulation of mRNA expression for METTL3 (0.2605-fold; p = 0.0007); METTL14 (0.4137-fold; p = 0.0068); WTAP (0.2740-fold; p = 0.0004); YTHDF1 (0.7170-fold; p = 0.0793); YTHDF2 (0.6269-fold; p = 0.0224); YTHDC1 (0.9867-fold; p = 0.0008); YTHDC2 (0.0570-fold; p = 0.0003); FTO (0.4534-fold; p = 0.0039); ALKBH5 (0.0066-fold; p = 0.0004); BDNF (0.0106-fold; p = 0.0006) and CREB (0.9172- fold; p = 0.0003). However, YTHDF3 (1.335-fold; p = 0.0647) was upregulated. FA increased protein expression of BDNF (1,205-fold; p = 0.0173) and P-CREB (1.5537-fold; p = 0.0002) and decreased protein expression of PI3K (0.8411-fold; p = 0.0346) and P-Akt (0.8274-fold; p = 0.0614). The observed increase in global m6A, despite downregulation of ‘writers’ and ‘erasers’ underscores a complex interplay of compensatory mechanisms resulting from FA exposure. The differential expression of m6A ‘readers’, particularly the upregulation of YTHDF3 and downregulation of YTHDF1/2 and YTHDC1/2, suggests selective stabilization of survival-related transcripts to counteract FA-induced neurotoxicity. FA exposure resulted in the upregulation of BDNF and P-CREB protein levels which indicates a compensatory mechanism aimed at preserving neuroprotective signalling despite transcriptional repression. PI3K and P-AKT were downregulated indicating a suppression of growth and survival pathways which are potentially linked to oxidative stress and energy conservation under toxic stress.
Thymoquinone triggers DNA hypomethylation in human colorectal adenocarcinoma (CACO-2) cells.
(2024) Mangerah, Aaliyah.; Chuturgoon, Anil Amichund.; Ghazi, Terisha.
DNA hypermethylation is a frequent feature of colorectal cancer, where it has been linked to the silencing of tumour suppressor genes and cancer progression. Thymoquinone (TQ) is a bioactive compound found in black cumin (Nigella sativa), and displays promising anti-cancer effects; however, its epigenetic effect in colorectal cancer is uncertain. This study investigated the impact of TQ on global DNA methylation in the human colorectal adenocarcinoma (Caco- 2) cell line. Caco-2 cells were cultured and treated with TQ for 24 hours. The MTT assay was conducted to assess cell viability and obtain an IC50. Global DNA methylation was quantified using an ELISA kit. Changes in mRNA expression of DNMT1, DNMT3a, DNMT3b, UHRF1, MBD2, TET1, TET2, TET3, and miR-29b were determined with qPCR. Changes in DNMT1,DNMT3a, DNMT3b, and MBD2 protein expression were assessed by Western blotting or ELISA. TQ dose-dependently decreased cell viability and yielded an IC50 of 504 μM. TQ brought about global DNA hypomethylation (p < 0.005) in Caco-2 cells. TQ reduced mRNA expression of DNMT1 (p < 0.05), DNMT3a (p < 0.05), and DNMT3b (p < 0.005), as well as UHRF1 (p < 0.05) and MBD2 (p < 0.05). TQ increased TET1 (p < 0.05), TET2 (p < 0.05), and TET3 mRNA expression (p < 0.005). MiR-29b expression was also increased (p < 0.05). TQalso reduced protein expression of DNMT1 (p < 0.0001), DNMT3a (p < 0.0001), DNMT3b (p< 0.05) and MBD2 (p < 0.005). Together, these results suggest that TQ induced global DNA hypomethylation in Caco-2 cells by down-regulating DNMT1, DNMT3a, DNMT3b, UHRF1,and MBD2, and up-regulating TET1-3 and miR-29b expression. This highlights the potential of TQ as a promising anti-cancer agent.
Fumonisin B1 induces oxidative stress, mitochondrial dysfunction, mitophagy revention and DNA hypermethylation in C57BL6 mice lung tissue.
(2024) Govender, Anthia Camara.; Chuturgoon, Anil Amichund.; Ghazi, Terisha.
Fumonisin B1 (FB1) is acknowledged as the most toxic variant of the Fusarium mycotoxins, largely due to its prevalence as a major naturally occurring fumonisin in agricultural products. The consumption of FB1 is associated with significant health risks for humans and animals. FB1 induces mitochondrial toxicity through the disruption of the mitochondrial electron transport chain (ETC), leading to mitochondrial membrane depolarization and an increase in reactive oxygen species (ROS) production. The aim of this investigation was to examine the mitochondrial toxicity in the lung tissue of mice treated with FB1 for 24 hours, along with the effects of FB1 on oxidative stress, mitophagy, and global DNA methylation. C57BL/6 mice (n=5/group) were orally administered 0.1 M phosphatebuffered saline (PBS) or 5mg/kg FB1 for 24 hours. Thereafter, the lungs were harvested, and RNA and protein were extracted. The TBARS assay was employed to measure lipid peroxidation. qPCR was used to corroborate the mRNA expression of oxidative stress-related genes [superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), nuclear factor erythroid 2-related factor 2 (Nrf2), catalase (CAT) and glutathione peroxidase (Gpx)], mitochondrial stress mitigating and mitophagy related genes [sirtuin 3 (Sirt3), Lon peptidase 1 (Lonp1), PTEN-induced kinase 1 (Pink1), sequestosome 1 (p62), and Parkin] and DNA methylation-related genes [DNMT1, DNMT3A,DN MT3B and methyl-CpG-binding domain (MBD2)]. Western blot was used to establish the protein expression of SOD2, CAT, Sirt3, Lonp1 and Parkin. Global DNA Methylation was assessed by ELISA. Malondialdehyde (MDA) was significantly upregulated (p<0.0001) in the lung tissue of FB1- treated mice. Further, there was a marked decrease in the expression of antioxidant defence-related genes, including Nrf2 (p<0.0001), SOD1 (p=0.0003), and Gpx (p=0.0004). Additionally, there was a notable decrease in both the gene (p<0.0001) and protein (p<0.0001) expression of CAT, while SOD2 gene (p=0.7454) and protein (p=0.7141) expression did not show significant variation in the lungs of the treated mice, when compared to the controls. In terms of the mitochondrial stress response, FB1 significantly increased Sirt3 transcripts (p=0.0244) and protein expression (p=0.0001), coupled with a significant decrease in Lonp1 gene (p<0.0001) and protein (p<0.0001) levels. Moreover, following FB1 treatment, significant reductions were observed in the expression of Pink1 (p<0.0001), Parkin(p =0.0162), and p62 (p<0.0001) genes, alongside a significant decrease in Parkin protein (p<0.0001) expression. Finally, a significant increase in both global DNA methylation (p=0.0018) and expression of DNMT3A (p=0.0082) and DNMT3B (p=0.0047) was noted; DNMT1 (p=0.1521) and MBD2 (p=0.6934) expressions showed no significant change in the lung tissue of FB1-treated mice, relative to controls. FB1 disrupted mitochondrial function and inhibited mitophagy in mouse lungs. Furthermore, it induced oxidative stress, that contributed to mitochondrial toxicity and global DNA hypermethylation.
Tetradenia riparia (hochst.) Codd (lamiaceae) aqueous leaf extract enhanced oxidant production and nf-b mediated apoptosis in hepatocellular carcinoma (HEPG2) cells.
(2024) Gcaba, Seluleko.; Khan, Rene Bernadette.; Khumalo, Hlolisile.
Introduction: Cancer has become a global health problem, contributing almost 20 million new cases and 10 million deaths worldwide in 2020. Ranked third globally, liver cancer accounted for 8.3% of the global cancer-associated mortality. Hepatocellular carcinoma (HCC) accounts for more than 90% of diagnosed primary liver cancer cases. Treatment may include chemotherapy, radiation therapy and liver transplant, but these treatments are expensive and induce serious adverse effects that reduce the efficacy of treatment. Medicinal plants are the suggested alternate treatment option attributed to anti-microbial, antioxidant and anti-inflammatory effects that may ameliorate the harsh side effects, but also demonstrate anti-cancer activity. Tetradenia riparia is a readily available South African shrub with medicinal and anti-cancer potential, but the mechanisms require elucidation. This study aimed to investigate the antioxidant and cell death mechanisms activated by Tetradenia riparia aqueous leaf extract (TRALE) in HCC (HepG2) cells. Methods: HepG2 cells were treated with several concentrations of TRALE (0–3000 μg/ml) for a duration of 24 hours to assess the cell viability. The methylthiazol tetrazolium (MTT) assay was used to obtain an IC50, which was used to treat the HepG2 cells for all subsequent assays. After treatment, mitochondrial activity was assessed as a measure of cell viability using luminometry for the ATP and mitochondrial membrane potential (m) assays. The cells were assayed for oxidative stress by quantifying free radical mediated membrane damage (TBARS, NOS and LDH cytotoxicity assays, and iNOS gene expression), and antioxidant response using luminometry (GSH, GSSG, ratio of GSH/GSSG), qPCR (SOD2, catalase, GPx1, Nrf2) and western blotting (HSP70). Induction of apoptosis was determined by luminometrically quantifying caspase-3/7, caspase-8, and caspase-9 activities. Furthermore, the Annexin V apoptosis and necrosis assay elucidated the cell death pathway. Protein expression of cIAP (Western blot) and qPCR for NF-κB and BCL-2 were also evaluated. Results: After a 24-hour exposure, TRALE conferred a dose-dependent reduction in cell viability that was associated with a notable drop in ATP at the IC50. An increase in ΔΨM led to increased levels of ROS, which increased lipid peroxidation and RNS production despite downregulated iNOS. Elevated SOD2 in response to ROS production enhanced the HepG2 cell’s ability to convert superoxide radicals into H2O2, while catalase and Gpx1 were upregulated to prevent harmful ROS formation and protect macromolecules from oxidative damage. Increased GPx1 was associated with depletion of GSH and the GSH/GSSG ratio, but Nrf2 was downregulated and HSP70 was similar to the control. The downregulation of Nrf2 was associated with increased NF-κB. Initiator caspase-8 activation corresponded with downregulated cIAP, while downregulated BCL-2 contributed to caspase-9 activation. In addition, activation of caspase-3/7 facilitated phosphatidylserine externalisation. Necrotic markers and LDH were decreased, suggesting that TRALE induced apoptosis in HepG2 cells. Conclusion: The decreased cell viability was associated with depletion of ATP, while m contributed to ROS production that increased RNS and caused lipid peroxidation. Although GSH was depleted and Nrf2 was downregulated, the upregulated SOD2, catalase and Gpx1 suggest a response suggested a response to ameliorate oxidative stress as implied by unchanged HSP70. However, oxidants were still elevated and associated with increased NF-κB. Interestingly, iNOS expression was downregulated, thus NF-B contributed to initiation of apoptosis that was associated with decreased BCL-2 and cIAP, and increased caspase activity. In addition, necrosis was not evident. Therefore, the TRALE-treated HepG2 cells were more susceptible to apoptosis. Further studies are required to elucidate the role of NF-κB in TRALE-induced apoptosis.
An in-silico and in-vivo investigation on the effects of acute fumonisin b1 exposure on inflammation and epigenetics in C57BL/6 mice hearts.
(2024) Gounder, Selwyn Kyle.; Chuturgoon, Anil Amichund.; Ghazi, Terisha.
Fumonisin B1 (FB1), a mycotoxin produced by Fusarium species, is a significant contaminant in cereal grains, posing serious health risks. This pilot study investigated the cardiotoxic effects of acute FB1 exposure on inflammation and epigenetic modifications in C57BL/6 mice. Given the pilot nature of this study, acute exposure was prioritised to establish initial findings. Molecular docking was employed to predict the binding interactions of FB1 with key inflammatory proteins, including tumour necrosis factor-alpha (TNF-α), inducible nitric oxide synthase (iNOS), and nuclear factor kappa B (NF-κB) subunits. In vivo experiments involved treating mice with 5 mg/kg FB1 for 24 hours, followed by heart tissue analysis using quantitative polymerase chain reaction (qPCR), Western blotting, nitric oxide synthase assay (NOS assay), and enzyme linked immunosorbent assay (ELISA) to assess selected gene and protein expression levels of inflammatory markers and DNA methylation. Docking results indicated that FB1 binds to inflammatory proteins TNF-α, iNOS, NF-κB (p65), and NF-κB (p50), potentially altering their function. Gene expression analysis revealed significant downregulation of pro-inflammatory cytokines (TNF-α, interleukin-6 (IL-6), interleukin-1β (IL-1β)) and the anti-inflammatory cytokine interleukin-10 (IL-10), while protein analysis showed an upregulation of these cytokines, suggesting a complex regulatory mechanism. Additionally, FB1 exposure led to increased levels of reactive nitrogen species and significant upregulation of DNA methylation, indicating epigenetic modulation. This study elucidates the cardiotoxic effects of FB1 on mice, emphasizing the intricate interplay between inflammatory pathways and DNA methylation. Molecular docking studies suggest that FB1 may bind to key residues on TNF-α, iNOS, and NF-κB subunits, potentially modulating these proteins' activity and triggering inflammatory responses. In vitro analysis demonstrated significant dysregulation of inflammatory and DNA ethylation-related genes, with a notable upregulation of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) and anti-inflammatory mediators (IL-10, Transforming growth factor beta 1 (TGF-β1)) at the protein level. The observed disparity between gene and protein expression could be attributed to several factors, including post-transcriptional and post-translational modifications. These modifications are crucial in biological processes and can cause differences between mRNA and protein levels. Translational regulation, which involves the recruitment of various mRNA species to the ribosome, can lead to a decreased correlation between mRNA and protein amounts. Additionally, the study found contradictory DNA methylation results: global DNA methylation levels were upregulated, indicating hypermethylation, while DNA methylation gene (DNMT) expression was decreased. This suggests a complex interplay between DNA methylation and gene expression, potentially influenced by other regulatory mechanisms like microRNAs. These findings highlight the need for further validation using additional tests, such as Northern blots and microarray assays. Overall, the study underscores FB1's ability to activate inflammatory pathways and cause cardiac distress through cytokine dysregulation and epigenetic changes. Further research is essential to fully understand the mechanisms of FB1- induced cardiotoxicity and the potential therapeutic role of DNA methylation in mitigating these effects.
Ursolic acid as a potential inhibitor of mycobacterium tuberculosis cytochrome bc1 oxidase: a molecular modelling perspective.
(2021) Tembe, Ntombikayise.; Mhlongo, Ndumiso Nhlakanipho.; Kumalo, Hezekiel Mathambo.
Tuberculosis (TB) is a disease, caused by an infectious agent; Mycobacterium tuberculosis, which persists as a major problem globally, especially in developing countries such as Brazil, Indonesia, and South Africa. Individuals who are diabetic and human immunodeficiency virus (HIV) co-infected are at a higher risk of contracting TB. Hence, these risk factors are associated with a compromised immune system. Among these factors, various strains are involved in the pathogenesis of TB such as multidrug-resistant tuberculosis (MDR-TB) and extensively drugresistant tuberculosis (XDR-TB) strains. The emergence of these strains may result from failure to complete treatment within the stipulated period of six months. However, studies show that the protein QcrB; contributes more to TB pathogenesis. Therefore, there is an urgent need for the discovery of drugs that inhibit QcrB. The current FDA-approved anti-tubercular drugs such as, Lansoprazole sulfide (LSPZ) and Telacebec (Q203) which inhibit QcrB are bacteriostatic and have been linked to side effects including dementia, chronic kidney disease, and ischemic cardiac diseases [1], thus prompting a search for an alternative drug. Various natural compounds have been reported to possess several bioactivities that could be crucial in the management of tuberculosis (TB) disease. Warbugia salutaris, a medicinal plant has been found to exhibit inhibitory properties against M. tuberculosis. Numerous compounds are derived from W. salutaris. In this study, we focus solely on Ursolic acid (UA) and its derivative, Ursolic acid acetate (UAA). These two compounds possess antibacterial, anti-HIV, and antimycobacterial properties. This suggests that they could potentially possess inhibitory properties towards M.tuberculosis QcrB protein. In this study, computational methods are applied to investigate the inhibitory activity of UA and UAA on M. tuberculosis QcrB. Molecular Docking, Molecular Dynamics (MD) simulations, Radius of Gyration, Principal Component Analysis (PCA), and Molecular Mechanics-Generalized Born Surface Area (MM/GBSA) binding free energy calculations were performed in explicit solvent to accomplish our goal. The obtained results indicated that the (1) the binding of UA to QcrB induced a more stable and compacted conformation compared to LSPZ and Q203; (2) high total binding free energy estimated in the QcrB-UA system was due to numerous hydrophobic residues in the binding site of QcrB that interact with phenyl rings of UA resulting in hydrophobic packing. This implies that UA has a high binding affinity and, as a result, a strong inhibition of QcrB; (3) more H-bonds were observed in the QcrB-UA system than in the QcrBQ203 system; (4) rigidity was displayed mostly in Arg124 and Thr128; (5) Arg124 and Phe127 also contributed more to the total binding energy in QcrB-UA and QcrB-UAA. This implies that the ligands exert a high binding affinity in the porphyrin binding site than in the active site. The identification of a molecule that competes with the porphyrin ring for the binding site could be beneficial in QcrB pharmacological research; (6) UA could be a potential anti-tubercular agent through QcrB inhibition, although it is hepatotoxic within tolerable concentrations. However, observed potential hepatotoxicity was based on predictions. Although the preliminary findings of this report warrant further experimental validation, they lay a strong foundation for subsequent assessment and development of these natural compounds as antitubercular drugs.
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.
N, N Bis (2-Pyridylmethyl)-1, 2-Ethylenediamine Tetrahydrochloride Stimulates Intrinsic Apoptosis Mediated by Oxidative and Nitrosative Stress Induction of the NF-B/STAT3 Pathway in Human Hepatocellular Carcinoma (HepG2) Cells.
(2022) Ntanzi, Nosipho.; Khan, Rene Bernadette.
Introduction: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Its incidence is rising, and this trend is expected to continue for decades. Several cancer therapeutics have already been discovered and are being used to treat HCC, however, most of them cause severe side effects which decrease the treatment's effectiveness. Metal chelators such as ethylenediaminetetraacetic acid (EDTA) have previously demonstrated anti-cancer potential. N, Nbis (2-pyridylmethyl)-ethylenediamine tetrahydrochloride (H2pmen) is a tetradentate ligand that forms stable complexes with Fe, Cr, Cu(II), and Zn (II), and it has been shown to be a potentially effective reagent for metal chelation. This study investigated the antiproliferative and cytotoxic effects of H2pmen in the HepG2 cell line. Methods: The cell viability was determined by treating HepG2 cells with different concentrations (0–1000 μM) of H2pmen over 24h. MTT assay was used to obtain an IC50, which was then used in all subsequent assays. The cells were then assayed for oxidative stress and membrane damage (TBARS, NOS, GSH, and LDH cytotoxicity), apoptotic induction (ATP assay, JC-10 assay, Annexin v, Caspases), cytochrome P450 3A4 activity (Luminometry). Protein expression of iNOS, SOD2, Bax, Caspase-2, and STAT3 were identified using western blot analysis. The gene expression of GPx1, Nrf2, NF-κB, p53, and OGG1 was determined using qPCR. Results: H2pmen induced a dose-dependent decrease in cell viability (IC50 of 209 μg/ml), a significant increase in CYP34A activity (p0.05 at IC20 and IC50), a decrease in ATP production (at IC20 p0.05 and at IC50), a significant decrease in m (p0.05 at IC20 and at IC50). The ROSassociated membrane was induced, indicated by an increase in lipid peroxidation (p0.05 at IC20 and p≥0.05 at IC50), an increase in RNS production (p≥0.05 at IC20 and at IC50), an upregulation in iNOS protein expression (at IC20 where p0.05 and at IC50) and NF-κB gene expression (at IC20 where p0.05 and at IC50). Oxidative stress occurred due to a decrease in GSH levels (at IC20 and p≥0.05 at IC50), a significant downregulation in SOD2 protein expression, and upregulation in gene expression of GPx-1 (at IC20 where p≥0.05 and at IC50) and Nrf2 (at IC20 and at IC50 where p0.05). H2pmen initiated caspase-dependent apoptosis that was indicated by a decrease in Caspase-2 (p0.05at IC20 and at IC50), caspase-8 (at IC20 and p≥0.05 at IC50), a slight insignificant decrease at IC20 and an increase at the IC50 in caspase-9, a significant upregulation in Bax (p0.05 at IC20 and at IC50) protein expression and p53 (at IC20 where p0.05 and at IC50) gene expression. The significant increase in caspase-3/7 (p≥0.05 at IC20 and IC50), Annexin V levels (p≥0.05 at IC20 and at IC50), LDH (p≥0.05 at xviii IC20 and IC50), STAT3 (p0.05at IC20 at IC50), PARP1 (p0.05 at IC20 and at IC50), and OGG1 (p0.05 at IC20 and at IC50) shows that apoptosis was executed by H2pmen in HepG2 cells. Conclusion: H2pmen reduced cell viability of HepG2 cells, exerting a cytotoxic effect associated with decreased m and ATP, and increased LDH leakage. The chelating properties of H2pmen was linked to the induction of oxidative and nitrosative stress that affected lipids and DNA. The HepG2 cells mounted an antioxidant defense involving Nrf2 to counteract the depletion of SOD2 and GSH, with evidence of its effect associated with upregulation of GPx. The prevailing oxidative stress activated DNA repair enzymes (PARP1 and p53), while NF-κB and STAT3 pathways were also induced. Bax-induced MOMP and caspase-2 invoked VDAC triggered caspase-dependant apoptosis via the intrinsic pathway.
Momordica foetida facilitates glucose uptake independent of AMPK2 and PI3K to attenuate hyperglycemia-induced oxidative stress via a JNK-STAT3 mediated pathway in HepG2 cells.
(2022) Netshitangani, Tshamano Fulufhelo.; Khan, Rene Bernadette.
Introduction: The exponential rise in the global prevalence and incidence of type 2 diabetes is concerning. Hyperglycemia is a hallmark of type 2 diabetes that induces oxidative stress, leading to impairment of vital liver metabolic pathways. Metformin is the first-line treatment for type 2 diabetes mellitus. However, Momordica foetida has been used in folk medicine for the treatment and management of diabetes mellitus in various parts of the world including South Africa. Aim: In the present study, the cytoprotective effects of M. foetida on liver impaired glucose metabolism and oxidative stress damage were investigated on high glucose induced HepG2 cells, with Metformin as a positive drug control. Methods: The M. foetida leaves were used to prepare an aqueous lyophilized extract. The HepG2 cells were serum starved for 1 hour, then exposed to hyperglycemic conditions (30mM D-glucose) for 24 hours. Cells were treated with various concentrations (125 - 1000 μg/ml) of the lyophilized M. foetida aqueous extract for 24 hours, and the 3-(4,5- dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT) assay evaluated the effects of high glucose and M. foetida on the metabolic activity of HepG2 cells. Antioxidants and prooxidants were assessed and quantified using luminometry, thiobarbituric acid reactive substances (TBARS) and nitric oxide synthase (NOS) assays. Western blot and quantitative real-time (qPCR) were used to observe the effects high glucose and M. foetida on signaling pathways and antioxidant response. Results: Glucose uptake in hyperglycemic conditions was mediated by increased gene expression of adenosine monophosphate-activated protein kinase alpha 2 (AMPKα2) (p˂0.05) and phosphatidylinositol 3‑kinase (PI3K) (p˂0.05), but glucose transporter 2 (GLUT2), glucokinase (GK) and glycogen synthase (GS) were downregulated (p˂0.05). Interestingly, an opposing response was noted for Metformin and M. foetida treatments, where AMPKα2 (p˂0.05) and PI3K (p˂0.05) were downregulated, whereas GLUT2, GK and GS were upregulated (p˂0.05) compared to the hyperglycemic control. When compared to the hyperglycemic conditions control, M. foetida treatments and Metformin showed an increase in glucose uptake. Hyperglycemic conditions induced toxicity indicated by increased extracellular lactate dehydrogenase (LDH) and decreased adenosine triphosphate (ATP), but Metformin and M. foetida decreased LDH activity back to xvi normoglycemic levels, indicating reduced cytotoxicity. Increased mitochondrial membrane potential (m) in hyperglycemic conditions was accompanied by increased lipid peroxidation (p˂0.05) and reactive nitrogen species (RNS) (p˂0.05). The m was increased further by M. foetida, with minimal effect on reactive oxygen species (ROS) production but effectively increasing RNS (p˂0.05). Oxidative damage was reduced in the hyperglycemic control but was increased by Metformin and M. foetida treatments prompting the activation of p53 in these cells (p˂0.05). Effective oxidative stress response was mounted by NRF2 (p˂0.05) and antioxidants SOD2 (p˂0.05) and GSH, but GPx1 and CAT (p˂0.05) were decreased. Interestingly, Metformin and M. foetida induced CAT (p˂0.05) and GPx1 (p˂0.05) in the antioxidant response, consequently decreasing GSH. Metformin decreased NRF2 (p˂0.05) and SOD2, while M. foetida increased NRF2 significantly and had no effect on SOD2 relative to the hyperglycemic control. Hyperglycemic conditions downregulated the oxidative stress response by MAPK (p-p38, pJNK and pERK1/2) (p˂0.05). However, Metformin upregulated pJNK (p˂0.05) and pERK1/2 (p˂0.05), but p-p38 (p˂0.05) was downregulated. Interestingly, M. foetida upregulated pJNK (p˂0.05), downregulated pERK1/2 (p˂0.05) and had no effect on p-p38. Hyperglycemic conditions also increased pSTAT3, which was downregulated by Metformin and M. foetida treatments (p˂0.05). Conclusion: Taken together, the results demonstrated that M. foetida enhanced the metabolic activity and reduced cell cytotoxicity in HepG2 cells. Furthermore, M. foetida facilitated glucose uptake independent of AMPK2 and PI3K. The main source of oxidative stress was increased RNS, which was alleviated by an effective MAPK/JNK and antioxidant response involving CAT.
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.
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.
Terminalia phanerophlebia crude aqueous leaf extract activates the NRF2-mediated antioxidant defence to prevent oxidative stress in human hepatocellular carcinoma cells.
(2021) Nyahada, Marcilyn Rutendo.; Khan, Rene Bernadette.
Abstract available in PDF.
The prevalence of Methicillin-resistant Staphylococcus aureus in blood product hampers at the South African National Blood Service.
(2021) Seoraj, Varsha.; Chuturgoon, Anil Amichund.; Singh, Ravesh.; Van den Berg, Karin.
Background: Methicillin-resistant Staphylococcus aureus (MRSA), are strains of the Gram-positive cocci known to cause various health conditions. Patients suffer from abscesses, skin infections and more severe conditions such as osteomyelitis and septicaemia. These bacteria are highly resistant to antibiotics and bacteria such as these are a great risk to the public, especially since Staphylococcus aureus is an opportunistic bacterium. In 2016, a donor unit received for the production of eye serum at South African National Blood Service (SANBS), when quality controlled, tested positive for MRSA. The bacterial contamination was traced to a staff member at the clinic where the blood was donated. Little research has been conducted to determine if MRSA is a problem and if it could negatively affect the blood supply. Aim: The aim of this study was to determine whether blood services contribute to the spread of MRSA and other bacterial pathogens through the blood product hamper system. Materials and Methods: A cross-sectional study to determine the prevalence of MRSA on 850 blood product hampers moving between SANBS inventory laboratories and blood banks, was conducted at SANBS between August 2020 and May 2021. Hampers were swabbed with a Sigma-Transwab containing liquid Amies transport medium for the detection of MRSA. The swabs were cultured onto CHROMagar MRSA where a rose or mauve coloured colony confirmed the presence of MRSA. Bacterial contaminants which were detected during the testing procedure were isolated, and loaded onto the Vitek 2 Compact for bacterial identification. Results: A total of 696 hampers were processed as per the study protocol (81.9%). Out of the 850 hampers planned to be swabbed, 143 (16.8%) hampers were not swabbed as a result of staff not performing the procedure and swabs from 11 hampers were omitted (1.3%) as they did not comply too protocol requirements. Of the 696 hampers swabbed, MRSA was not detected (0%) however, bacterial growth other than MRSA was observed. The most common isolates detected were Aerococcus viridans, Rothia dentocariosa, followed by Bacillis spp as well as Stenotrophomonas maltophilia. Conclusion: The study findings have shown that an effective hamper cleaning system is needed to safeguard the integrity of our blood supply. The findings of this study should be taken into consideration throughout all provinces at SANBS, for the consistent and regular cleaning of hampers, which carry blood and blood products.
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).
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.

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