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Masters Degrees (Medical Biochemistry)

Permanent URI for this collectionhttps://hdl.handle.net/10413/7039

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Browsing Masters Degrees (Medical Biochemistry) by Subject "Apoptosis."

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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.
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    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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    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.
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    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.
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