Khan, Rene.Kumalo, Hezekiel Mathambo.Naicker, Mayanka.2026-03-242026-03-2420232023https://hdl.handle.net/10413/24334Masters Degree. University of KwaZulu-Natal, Durban.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.enAdenosine triphosphate.Butylated hydroxytoluene.Complete culture medium.Colorectal cancer.Copper superoxide dismutase.Thesis