Khan , Rene.Kumalo, Hezekiel Mathambo.Nkambule, Awande Comfort Monwabisi.2026-03-242026-03-2420242024https://hdl.handle.net/10413/24337Masters Degree. University of KwaZulu-Natal, Durban.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.enMicrometer.Dimethyl sulphoxide.Fas ligand.Hydrogen peroxide.Optical density.Thesis