1,720,979 research outputs found
Evaluation of the neurotoxicity of pentachlorophenol and its active metabolites on SH-SY5Y neuroblastoma cells
Get PDFDissertation (MSc)--University of Pretoria, 2017.Pentachlorophenol (PCP) is an organochloride pesticide that is ubiquitous within the
environment due to its chemical stability. It is classified as a persistent organic
pollutant, and has been predominantly used in the wood preservation industry.
Workers and populations living close to PCP usage and production are exposed to it
via inhalation and dermal absorption, and ingestion of contaminated food and water.
It is lipophilic and is able to accumulate within various bodily systems, including the
brain. Adverse effects of PCP have been reported to varying degrees in the immune,
hepatic, and endocrine systems. Although neurological symptoms have been
associated with PCP exposure, knowledge of mechanisms of neurotoxicity is limited.
Elucidation of molecular mechanisms at a cellular level within neuronal cells is
required to contribute toward the current gap in the knowledge of PCP neurotoxicity.
The aim of the study was to evaluate the effects of PCP and its active metabolites,
tetrachloro-1,4-benzoquinone (TCBQ) and tetrachlorohydroquinone (TCHQ) in
human neuroblastoma SH-SY5Y cells.
Effects on cell proliferation were assessed using the sulforhodamine B (SRB) assay.
Flow cytometric analysis was employed to investigate effects on cell cycle using
propidium iodide (PI), mode of cell death using Annexin V-FITC and PI, reactive
oxygen species (ROS) using dichlorofluorescein, and mitochondrial membrane
potential (Δᴪm) using JC-1 fluorescence. Caspase-3 activity was assessed with Ac-
DEVD-AMC, and glutathione (GSH) with monochlorobimane fluorescence. Effects on
acetylcholinesterase (AChE) were assessed in vitro using the Ellman esterase
assay, as well as in silico via molecular docking and molecular dynamics simulation.
The IC50 concentrations of PCP, TCBQ and TCHQ were 80.0, 35.4, and 63.7 μM,
respectively. Cell cycle disruptions were revealed in the form of a G1 block and a
G2/M block as a result of PCP and TCHQ exposure, respectively, while TCBQ
resulted in a prolonged S phase traverse. The predominant mode of cell death of
PCP was necrosis, while TCBQ induced apoptosis. Exposure to TCHQ resulted in
one of two fates, being either predominantly apoptotic or necrotic cell death.
Decreased Δᴪm was an early event for all compounds, however, differed in their
involvement of inducing ROS. Oxidative stress was an evident mechanism of PCP
and TCBQ toxicity, as increased ROS was accompanied by lowered GSH, while reductive stress leading to subsequent oxidative stress was indicated by increased
ROS and GSH for TCHQ. All compounds yielded increased caspase-3 activity. The
fate of TCHQ exposed cells was postulated as a switch from apoptosis to necrosis
due to overwhelming ROS insult on apoptotic machinery, surpassing a threshold for
apoptosis capability. Inhibition of AChE was observed by only TCHQ in vitro, the
Ellman IC50 of which was 79.7 μM. In silico assessment supported a hypothesis of
TCHQ inhibition of AChE, with TCHQ-acetate bound ligands binding AChE receptors
with binding energies corresponding to the Ellman IC50. Binding stability was
confirmed by molecular dynamics.
Pentachlorophenol and its active metabolites exhibited different mechanisms of
toxicity toward neuronal cells, leading to different modes of cell death. A new
hypothesis for the molecular mechanism of TCHQ AChE inhibition was developed,
and sets a platform for further investigation.PhysiologyMScUnrestricte
Differential effects of Sutherlandia frutescens subs. microphylla on cell numbers, morphology, gene and protein expression in a breast adenocarcinoma and a normal breast epithelial cell line
No full textDissertation (MSc)--University of Pretoria, 2007.Sutherlandia frutescens is a South African herbal remedy traditionally used for various ailments and lately to improve the overall health in cancer and HIV/AIDS patients. Relatively little is known about the mechanisms of action of the constituents present in S. frutescens. The aim of this project was to examine the in vitro influence of crude ethanolic S. frutescens extracts in human breast adenocarcinoma (MCF-7) and non-tumorigenic breast epithelial (MCF-12A) cells after 48 h of exposure. Dose-dependent studies were conducted on cell numbers and metabolic activity by means of spectrophotometry. Morphological changes were determined with light-, fluorescent- and transmission electron microscopy (TEM). Cell cycle progression and apoptosis were analyzed using flow cytometry. The differential effects of S. frutescens extracts on gene expression levels in both the MCF-7 and MCF-12A cells were conducted utilizing micro array analysis. mTOR kinase activity was measured with an ELISA assay. S. frutescens reduced cell proliferation in both the non-tumorigenic MCF-12A and the tumorigenic MCF-7 cell line in a dose-dependent manner. The tumorigenic MCF-7 cells were more susceptible to S. frutescens treatment compared to the non-tumorigenic MCF-12A cells. Morphological characteristics of apoptosis and autophagy, including cytoplasmic shrinking, membrane blebbing and an increase in autophagic vacuoles were observed in both cell lines with the MCF-7 cells being more susceptible to autophagy and the MCF-12A cells less susceptible to autophagy and apoptotic cell death. TEM confirmed ultrastructural characteristics of autophagy in both cell lines. Flow cytometry revealed a G2/M arrest with no increase in apoptosis in MCF-7 cells and a G2/M arrest with an increase in apoptosis in MCF-12A cells treated with 1.5mg/ml S. frutescens extract. Microarray analyses revealed 325 statistically significantly differentially expressed genes in MCF-7 cells and 1467 genes in MCF-12A cells. The majority of S. frutescens-treated genes were down-regulated when compared to the vehicle-treated control in both cell lines. Several genes involved in DNA replication and repair were differentially expressed in response to S. frutescens exposure. These include Poly (ADP-ribose) polymerase family, member 2 (PARP-2) (down-regulated in both cell lines), PCNA (down-regulated in MCF-7 cells) and growth arrest and DNA-damage-inducible beta (GADD45B) (up¬regulated in MCF-12A cells). This suggests that abrogated expression of genes involved in DNA replication and repair play a role in inducing a G2/M cell cycle arrest in S. frutescens-treated cells. ELISA analysis of the mTOR kinase revealed a decrease in mTOR kinase activity in both cell lines after S. frutescens exposure. Therefore, attenuated mTOR kinase activity as a result of S. frutescens treatment in both cell lines is regarded as a central mediator in inducing autophagy suppressing gene expression and inhibiting ribosome biogenesis. Understanding of in vitro molecular mechanisms of S. frutescens enables researchers to focus on affected cellular mechanisms and identify active compounds with subsequent evaluation as possible candidates for use in anticancer therapy. The current study contributes to the unraveling of the in vitro molecular mechanisms and signal transduction associated with 70% ethanolic S. frutescens extracts, providing a basis for further research on this multi-purpose medicinal plant in Southern Africa.Physiologyunrestricte
In vitro effects of Sutherlandia frutescens extracts in human breast adenocarcinoma and normal breast epithelial cells.
No full textPoster presented at the University of Pretoria Health Sciences Faculty Day, August 2007, Pretoria, South Africa.Sutherlandia frutescens is a South African herbal remedy traditionally used for stomach problems, diabetes and lately to improve the overall health in cancer and HIV/AIDS patients. The in vitro influence of crude S. frutescens extract was investigated in human breast adenocarcinoma (MCF-7) and normal non-tumorigenic breast epithelial (MCF-12A) cells after 48 h of exposure to S. frutescens extracts. Dose-dependent studies (1.0-2.0mg/ml) were conducted on cell numbers and viability by means of spectrophotometry. Morphological changes were determined with fluorescent and transmission electron microscopy (TEM). Cell cycle progression and apoptosis were analysed using flow cytometry and Agilent’s Human-1A Oligo Microarray slides with 20,173 known human 60-mer oligonucleotide probes were employed to study the differential effects of S. frutescens extracts on gene expression levels in both the MCF-7 and MCF-12A cells. A statistically significant decrease to 50% of malignant cell numbers was observed after 48 h of exposure to 1.0mg/ml S. frutescens extract in MCF-7 cells and 1.5mg/ml in MCF-12A. 48 h exposure to 1.5mg/ml S. frutescens revealed 21% viable MCF-7 and 78% viable MCF-12A cells compared to vehicle-treated control cells. Morphological characteristics of apoptosis and autophagy, including cytoplasmic shrinking, membrane blebbing and an increase in autophagic vacuoles were observed in both cell lines with the MCF-7 cells being more susceptible to autophagy and the MCF-12A cells more susceptible to apoptotic cell death. TEM confirmed ultrastructural characteristics of autophagy in both cell lines. Flow cytometry revealed a G2/M arrest with no increase in apoptosis in MCF-7 cells and a G2/M arrest with an increase in apoptosis in MCF-12A cells treated with 1.5mg/ml S. frutescens extract. Microarray analysis revealed differentially expressed genes involved in cell cycle progression including proliferating cell nuclear antigen (PCNA) and poly (ADP-ribose) polymerase (PARP2) in MCF-7 and topoisomerase I (TOPM1), DNA polymerase-sigma (POLS) in MCF-12A cells. This study contributes to the understanding of molecular mechanisms and cell signaling events associated with in vitro anticancer responses of S. frutescens, thus enabling researchers to focus on affected cellular mechanisms to identify and characterize active compounds from these extracts with subsequent evaluation as possible candidates for use in anticancer therapy
In vitro study of in silico designed sirtuin 1 and bromodomain 4 inhibitors on human neuroblastoma SH-SY5Y and acute myeloid leukemia U937 cells
Get PDFDissertation (MSc)--University of Pretoria, 2018.In this novel study, the effects of newly in silico designed bromodomain 4 and sirtuin 1 inhibitors on cellular growth and death was investigated in the neuroblastoma SH-SY5Y and acute myeloid leukemia U937 cell lines. The investigated bromodomain 4 compounds were found to be not as potent when compared to the sirtiun 1 inhibitor, W137. Therefore, the anti-proliferative effects and mechanistic action of W137 on the two human cancer cell lines were investigated.
The regulation of cancer associated gene expression may be possible in SIRT1-advantaged cancers such as in neuroblastoma and acute myeloid leukemia through the inhibition of SIRT1, which functions by deactivating or down-regulating anti-cancer proteins through deacetylation. Crystal violet DNA staining was applied to study the effect of W137 on cell numbers of SH-SY5Y cells and inhibition of cell proliferation was achieved in a concentration dependent manner. An IC50(SIRT1) value of 20 µM for the SH-SY5Y cells and 25 µM for U937 cells was determined after 48 h exposure. The IC50(SIRT1) values obtained for the compound as tested on the two cell lines were comparable to other well-known SIRT 1 and 2 inhibitors and low enough to merit further experimentation. Qualitative studies on cell morphology employing fluorescent microscopy and triple dye staining indicated a decrease in cell density and loss of cellular membrane integrity. A slight increase in propidium iodide staining of DNA after 48 h exposure to the W137 compound suggested either necrosis and/or late stages of apoptosis.
Employing flow cytometry and propidium iodide to study the progression of the cell cycle revealed increased cell numbers in the sub-G1 phase in SH-SY5Y and U937 cells, suggesting cell death is induced by W137 after 24 and 48 h respectively. No significant cell cycle block in either the G1 or G2/M phase was observed, suggesting that the compound does not work by modulating the cycle in SH-SY5Y cells. The efficacy of W137 does not vary significantly after 24 h exposure compared to 48 h exposure for the SH-SY5Y cell line, while an increase in compound efficacy seems to be observed after an extended exposure period (48 h) for the U937 cell line.
Flow cytometry employing propidium iodide in conjunction with Annexin V was used to analyse cell death induced via apoptosis and/or necrosis and 24 h as well as 48 h exposure to the W137 compound revealed an increase in early and late apoptosis with a slight increase in necrosis, in SH-SY5Y cells. Analysis of the U937 cell line indicated a slight population shift towards early apoptosis after 24 h exposure and a slight shift towards late apoptosis and necrosis after 48 h. These findings confirm that cell death occurs via apoptosis in the SH-SY5Y as well as in the U937 cell line when exposed to W137. SH-SY5Y (Control: 22.32% vs exposed: 26.53%) and U937 (Control: 24.52% vs exposed: 43.07%) cells exhibited increased mitochondrial membrane depolarization and therefore the intrinsic mitochondrial pathway is the likely means by which apoptosis is achieved. Therefore, apoptosis is the mechanism by which cell death is achieved in both cell lines, as confirmed by cell cycle studies, analyses of cell death and mitochondrial membrane permeability studies in both cell lines. Levels of hydrogen peroxide (H2O2) generated within W137-exposed cells after 48 h was measured using DH2CF-DA and flow cytometry. After 48 h exposure, a statistically significant decrease in DCF-fluorescence SH-SY5Y cells (Control: 40 vs exposed: 13.4) but not U937 cells (Control: 251.4 vs exposed: 259.8) was observed, suggesting that increased ROS levels was not induced in response to W137 exposure after 48 h.
To study the effect of exposure to the W137 compound on the expression of the p53 and C-MYC genes, both key factors in healthy and cancerous cells, the quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) technique was employed. The SH-SY5Y cell line indicated a decrease in C-MYC levels upon Actinomycin D and W137 exposure after 24 h, while expression of the p53 gene was decreased after 24 h Actinomycin D exposure. Exposure to W137, however, increased p53 expression after 24 h as well as 48 h. p53 is crucial in programming cells for death and probably induces SH-SY5Y cells to enter apoptosis. Significant increase in C-MYC gene expression was observed in U937 cells after Actinomycin D exposure after 24 h and 48 h (p-value <0.05). Repression of SIRT1 therefore possibly resulted in the activation or increased expression of the pro-apoptotic gene p53, possibly enhancing cancer cell susceptibility to death and repair mechanisms. MYC oncoproteins, which are commonly up-regulated in human cancers of different organ origins, exert oncogenic effects by modulating gene and protein expression.
ELISA (Enzyme-linked Immunosorbent Assay) was employed to test for change in p53 protein K382 acetylation and activity following 48 h exposure to W137. The active, acetylated p53 protein is a crucial component in cell maintenance, damage detection and programmed cell death, and inhibition or down-regulation of p53 has been detected in many cancer types (1-3). Deacetylation of p53 by SIRT1 results in protein deactivation or down-regulation and therefore SIRT1 inhibition is expected to result in p53 being in the acetylated stage and active (4). Following 48 h exposure, analyses of the SH-SY5Y cells indicated a small but statistically significant increase in the expression of K382 acetylated p53 protein in Actinomycin D (1.17-fold) and W137 exposed cells (1.12-fold) (p-value <0.05).
In conclusion, the novel dual SIRT1 and 2 inhibitor W137 therefore inhibited cell proliferation in both the U937 and SH-SY5Y cell lines in a dose-dependent manner through the inhibition of cell growth and the induction of cell death in vitro. The two cell lines exhibited different reactions to the compound in some experiments but similar reactions in others, motivating the importance of further study into the cell line specificity and mechanistic variation of W137. The confirmation of these results by that obtained from the fluorescent microscopy study leads to the conclusion that apoptosis is the most likely cause of cell death for these cell lines. The current study contributes to the unravelling of the in vitro molecular mechanisms associated with and influenced by SIRT1 and SRT2 providing a basis for further research on this multi-functional cellular component and its diverse role in disease regulation. The W137 compound could potentially be an effective component in a drug designed to treat more than one type of cancer.
Keywords: Anti-cancer, acute myeloid leukemia (AML), neuroblastoma, sirtuin 1 (SIRT1), U937, SH-SY5Y, flow cytometry, apoptosis, cell cycle, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), gene expression, qRT-PCR, protein acetylation, ELISA, p53, C-MYC.NRF (THUTHUKA) fundingPhysiologyMScUnrestricte
Evaluation of a strategy based on multi-drug targeting of cancer proteins in breast cancer cell lines
Get PDFThesis (PhD)--University of Pretoria, 2020.Therapeutic inefficacy of conventional cancer treatment is a particular dilemma associated with metastatic triple negative breast cancer (TNBC), with patients still facing poor prognosis. The design and development of novel anticancer agents specifically targeted to cancer-associated pathways is of therapeutic interest. The rationale is twofold: firstly, targeted therapy overcomes widespread toxicity and adverse effects of conventional chemotherapy due to the selectivity of the treatment modality. Secondly, synergistic combinations of different classes of highly targeted therapies could hold therapeutic promise to overcome resistance by simultaneously circumventing multiple cancer hallmarks. This study evaluates the in vitro antiproliferative activity of six compounds using breast cancer cell lines as experimental model. Five of these compounds are novel, agents designed in silico to selectively target cancer hallmarks via inhibition of specific cancer-associated proteins. The compounds include an antimitotic (STX1972), three variants of bromodomain 4 (BRD4) inhibitors (Bzt-W41, Bzt-W49 and Bzt-W52), an inhibitor of both sirtuin (SIRT) 1 and 2 (W137) and an inhibitor of janus kinases 1 and 2 (Ruxolitinib). The synergism between paired combinations was also explored.
Two breast cancer cell lines, MDA-MB-231 and MCF-7 were used as experimental models. The MDA-MB-231 cell line is oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) negative and is therefore commonly used to model triple negative breast cancer with invasive and metastatic properties. MCF-7 cells are ER and PR positive and represent the hormone-dependent breast cancer model. The endothelial EA.hy926 cell line was used to represent non-cancerous cells. A crystal violet assay was used to determine the half maximal inhibitory concentration (IC50) of the six compounds on the tested cell lines after 48 h exposure. Drug combination studies based on the Chou-Talalay method of paired drug combinations were performed. Effects of treatment on cell morphology was assessed by means of confocal-microscopy. Flow cytometry was used to study the effects on cell cycle progression, apoptosis, autophagy/lysosomal activity, reactive oxygen species (ROS) production, changes in mitochondrial membrane potential (ΔΨm) and the serine 70 phosphorylation status of Bcl-2. Real-time quantitative PCR was used to analyse the effects of the compounds on the mRNA expression levels of p53, c-myc and bcl-2. Quantitative protein expression of c-MYC was analysed by means of enzyme-linked immunosorbent assay.
In vitro screening for antiproliferative activity revealed that the compounds showed cancer-selective cytotoxic effects when compared to the EA.hy926 control cell line. The initial screening identified three compounds for further investigative inclusion, namely the antimitotic (STX1972), the BRD4i (Bzt-W41) and the SIRTi (W137). STX1972 was found to inhibit cell growth in the nanomolar concentration range, whilst the rest of the compounds showed growth inhibition in micromolar concentration ranges. Bzt-W41 showed significant preferential selectivity for the TNBC MDA-MB-231 cell line versus the hormone-dependent MCF-7 cell line, while STX1972 and W137 exhibited only slight differential selectivity. Two combinations (STX + Bzt-W41 and Bzt-W41 + W137) exhibited synergism, whilst the STX + W137 combination exhibited antagonistic interaction. Cell cycle and apoptosis analysis revealed that STX1972 and Bzt-W41, alone and in combination, selectively induced cell cycle arrest and apoptosis in cancer cells. However, the W137 +Bzt-W137 combination did not show preferential targeting of breast cancer cell lines, with apoptosis induced equally or even more so in the control EA.hy926 cell line. STX1972 and Bzt-W41, as well as their paired combination, was further probed in aim of deciphering their individual and combined mode of action.
STX1972, Bzt-W41 as well as the paired combination proved to selectively inhibit cancer targets resulting in several molecular changes, leading to downstream pathway activation which culminates in both apoptotic and autophagy-related cellular demise. The study contributed towards deducing possible hypotheses regarding the mechanistic behaviours of the individual compounds and elucidated their combined effect during dual treatment. Results warrant future studies to further probe the intricate interaction of pathways involved in the synergistic combination of antimitotics and epigenetic regulators as a novel anticancer therapeutic modality.PhysiologyPhDUnrestricte
Proteomic characterisation of primary breast tumour extracellular matrix
Get PDFThesis (PhD (Pharmacology))--University of Pretoria, 2021.Breast cancer is the most commonly diagnosed cancer in women and is the leading cause of female cancer mortality worldwide. High cancer mortality rates, mostly due to late-stage diagnosis and the lack of appropriate personalised therapy, highlights treatment failure that prompts the need for continued research to identify new and improved breast cancer detection methods and treatment. The purpose of this study was to use advanced mass spectrometry-based proteomics to characterise and compare the proteome but especially extracellular matrix (ECM) protein components from solid invasive ductal carcinoma tumours to matched non-tumorous breast tissue with the aim of identifying potential prognostic markers or new therapeutic drug targets for breast cancer treatment.
Breast tumours are dense, complex tissue masses made up from a number of different proliferating cell types that are embedded in an intricate tumour microenvironment. Several studies have highlighted the role of the tumour microenvironment, more specifically the ECM, in tumour development and progression from localised invasion to advanced metastasis. The ECM consists of numerous protein components that provide a scaffold for both cell and growth factor binding, where ECM changes have been associated with tumour advancement. By implication, characterisation of tumour ECM components can potentially be used as prognostic or staging markers for breast cancer or to identify new targets for anticancer therapies.
In this research study, cryotome cut slices of snap-frozen tumour biopsies resected from patients diagnosed with invasive ductal carcinoma (IDC) were used to characterise the primary breast tumour proteome especially for the ECM. Haematoxylin and eosin staining, the gold standard for routine histopathological diagnosis of cancer, was used to visualise tissue morphology and to confirm the clinical IDC diagnosis.
An optimised protein extraction method involving high pressure cycling technology was used for tissue homogenisation and protein solubilisation of tumour biopsies. Proteomics analysis involving innovative semi-automated and cutting-edge sample preparation and liquid chromatography tandem mass spectrometry-based methods that are at the fore-front of drug target validation, drug discovery and prognostic marker identification, were used to acquire proteomic data from the tissue isolated from both tumour biopsies and equivalent non-tumorous breast tissue. A semi-automated sample preparation method using hydrophilic affinity-based protein capture, clean-up and off-bead trypsin digestion was used to produce peptides, followed by analysis using a Dionex Ultimate 3000 RSLC system coupled to an SCIEX 6600 TripleTOF mass spectrometer. Data independent acquisition using sequential window acquisition of all theoretical mass spectra (SWATH) data was collected and bioinformatic protein identification and relative quantification was performed. SWATH data provided reliable proteomic assessment, could identify low abundance proteins as well as provide relative quantitation of differentially expressed proteins in tumour samples. Tumour associated ECM changes were classified through STRING pathway analysis comparing the relative protein abundance between non-tumorous and tumour masses.
Pathway analysis revealed that ribosomal, spliceosome and endoplasmic reticulum protein processing pathways with associated protein components were significantly upregulated in breast tumour samples. Proteomic data confirmed that protein homeostasis, associated with protein synthesis, protein folding and alternative splicing, is severely affected in solid tumours in order to meet the demands of uncontrolled tumour growth and promotion of tumour metastasis. SWATH-based quantification and pathway enrichment analysis did identify several ECM protein networks containing a number of differentially expressed ECM proteins in breast tumour samples. These ECM proteins within the tumour microenvironment are involved in several cancer related biological processes that include structural integrity, cancer cell proliferation, tumour growth, tumour tissue invasion, and metastasis. These differentially expressed ECM proteins could potentially be used as putative biological prognostic signatures for breast cancer or be used as new drug targets to slow or completely inhibit breast cancer advancement and progression.
This exploratory study provides valuable proteomic data for breast cancer research associated with the tumour microenvironment and has laid the foundation for prognostic and pharmacological based studies for cancer therapeutics by identifying putative ECM protein candidates that can be further assessed in independent verification and validation breast cancer studies.PharmacologyPhD (Pharmacology)Unrestricte
Characterising select hepatocyte cultures for improved hepatotoxicity testing
No full textDissertation (MSc (Pharmacology))--University of Pretoria, 2023.Drug-induced hepatotoxicity is a major contributor towards post-marketed drug withdrawals. Most of these liver injuries can be associated with drug metabolism, which is primarily performed by hepatic cytochrome P450 enzymes, a superfamily of drug metabolising haem enzymes. Pre-clinical in vitro models are commonly used in an attempt to predict the toxicity profile of lead drug compounds during the early development phases. These in vitro models need to accurately resemble the human liver functionality to be able to predict drug hepatotoxicity. Primary human hepatocytes are the ‘gold standard’ for mimicking liver function. However, due to their expensive culturing requirements, limited time in active culture, and limited availability, other commercially available, transformed hepatic cell lines are commonly used as substitutes for primary hepatocyte cultures. The HepG2 cell line is one of the most commonly used because they are readily available, and the culturing methods are relatively inexpensive. Conventional monolayer culturing of human-derived cell lines has shown limitations when used for drug toxicity tests and lacks sufficient resemblance to human liver tissue, compared to three-dimensional cultured cells. This has led to three-dimensional cell cultures being recommended over monolayer cultures to predict potential in vivo toxicity.
The aim of this study was to characterise HepG2 cells differentially cultured as monolayer or three-dimensional spheroids, in the presence and absence of chronic enzyme-inducing drug cocktail exposure. Also, to evaluate the models’ feasibility over an extended culture time and compare their metabolic capabilities as candidates for hepatotoxicity screening platforms. This was done by generating HepG2 spheroids using the liquid overlay method for up to 21 days and culturing same origin HepG2 monolayers for 17 days. Cells were evaluated for morphology, viability, protein content, monolayer cell cycle profile, proteins mass profile differences, and the presence of hepatic markers in spheroids. The metabolic activity was assessed using liquid chromatography tandem mass spectrometry.
The monolayer and drug cocktail exposed monolayer cells were viable for up to 17 days while the spheroids and drug cocktail exposed spheroids were viable up to 21 days. CYP1A2 activity was detected in all cultures with slightly more acetaminophen (μmol/μgprotein) detected in monolayer cultures. The activity confirms CYP1A2 expression detected in all spheroid cultures from Day 7 to Day 21. Furthermore, minimal hydroxybupropion, dextrorphan and hydroxymidazolam was detected in all cultures suggesting low CYP2B6, CYP2D6 and CYP3A4 activity, respectively. The metabolite levels were similar between induction and non-induction cultures suggesting that the induction drug cocktail had no significant effect on the metabolic capacity of the HepG2 cells under either of the culturing conditions used. Therefore, the metabolic activity may be due to accumulated innate metabolic capability and/or long-term culture.
Furthermore, the growth plateau observed in spheroid cultures’ protein levels after Day 4 and the hepatic markers (AFP, HNF-4α, CK18 and albumin) expression observed from Day 7, would be desirable for repeated hepatotoxicity testing. Other studies have shown HepG2 spheroid cell viability and increased hepatic marker expression for more than 21 days, with a relatively consistent metabolic profile after 21 days, suggesting a stable differentiated phenotype. This is beneficial for repeated, long-term drug exposure for acute and chronic hepatotoxicity screening.National Research Foundation Innovation Master’s Scholarship (grant number:120972)Roche Products bursary (2021)PharmacologyMSc (Pharmacology)UnrestrictedFaculty of Health SciencesSDG-03:Good heatlh and well-bein
Assessing the in vitro efficacy of in silico designed compounds targeting the malarial Qi site of cytochrome bc1
Get PDFDissertation (MSc)--University of Pretoria, 2018.Plasmodium falciparum is the causative agent of the most commonly fatal form of malaria in Africa with annual deaths of more than 300 000. The rapid development and spread of antimalarial drug resistance by the parasite have stimulated research into the development of new drug classes. Target-based drug discovery have been used as a prominent and efficient tool to identify lead drugs. Reports suggest that selectively inhibiting the parasite mitochondrial electron transport could be a potential treatment effective at multi-stages of the parasite life cycle. Inhibitors of cytochrome bc1 (Cyt bc1), an essential inner mitochondrial membrane protein that drives ATP synthesis in the mitochondria are claimed to be lethal to apicomplexan species including Plasmodium. The emergence of resistance to atovaquone, a Cyt bc1 complex Qo site inhibitor, casts doubt over the long-term efficacy of new drugs targeting these mitochondrial proteins. Many aspects of potential drugs must be investigated to assess the suitability of new emerging drugs targeting the mitochondrion.
In silico target-based drug design methods using Autodock vina were used to design compounds that would theoretically bind to and inhibit the Qi site of Cyt bc1 of the P. falciparum. The potential candidate compounds were selected from compounds defined by Gamo et al., (2010) and tested using in silico docking experiments. Homology models were developed and modified to improve their drug-likeness according to the Lipinski rule, QED parameters and synthesised by Wuxi App Tec. This study assessed the antiproliferative activity of six candidate compounds on P. falciparum parasites in vitro following in silico compound docking and drug likeness assessment. Initial in vitro screening data was obtained for the test compounds at 1 and 5 ?M over 96 h and full dose-response curves was performed for compounds showing >70% proliferation inhibition at 1 ?M against the 3D7 strain. Four of the test compounds, EE1, EE3, EE5 and EE7 gave IC50 values of 89 nM, 664 nM, 64 nM and 249 nM, respectively. The candidate compounds had a marginal >2-fold selectivity towards malaria parasites but did not show cross resistance, with resistance indices of >120. In conclusion, in silico docking using software programs could be utilised as a potential tool for rapidly identifying feasible target-based antimalarial compounds while avoiding high throughput screening. Other possible target sites on the mitochondrion can be used to design new chemotypes. All the designed compounds showed significant antimalarial activity against the asexual stages tested on 3D7 strain with a significant resistance index. However, these compounds showed minimal activity on the gametocyte stage. Finally, compound EE5 showed to be the most potent, more selective and with higher resistance index, hence this can be further optimised for preclinical studies.PharmacologyMS
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
- …
