Abstract
Globally, breast cancer affects a significant percentage of women, making it a major public health concern. It accounts for 16 % of all cancers diagnosed in females and is the leading cause of cancer incidence and death, surpassing cervical cancer. In 2020, there were 2.3 million women diagnosed with breast cancer, resulting in 685 000 deaths globally. Although it primarily affects women, approximately 0.5 % to 1 % of men are also diagnosed with breast cancer. The current cancer treatments often result in severe negative side effects for patients and are often not readily available in rural settings. It is thus important to investigate novel, potential, safe, and cost-effective anti-cancer treatments. Agathosma betulina (A. betulina) is a plant used medicinally for its various health benefits and has been tested for its anti-inflammatory, antimicrobial, and antioxidant properties. However, the understanding of A. betulina’s potential as an anti-cancer agent is not thoroughly studied. The aim of the study is to investigate how A. betulina extracts induce cell death in breast cancer cells. The dried leaves of A. betulina were collected, ground into a fine powder, and extracted in sequence using ethyl acetate and methanol. The cytotoxicity of the crude extracts, methanol and ethyl acetate, was determined through the use of Alamar Blue reagent on breast (MCF-7) cancerous cells and lung (MRC-5) normal cells. The adenosine triphosphate (ATP) bioluminescence assay was quantitatively used to assess the ATP levels in half maximum inhibitory concentration (IC50)-treated cancerous cells. Lactate dehydrogenase (LDH) was employed to measure the damage caused to the MCF-7 cells by the extracts in order to determine the mechanism of the triggered type of cell death. Fluorescence and brightfield microscopy were utilised to analyse nuclear and morphological changes, respectively, in cancer-treated cells. The Thiobarbituric Acid Reactive Substances (TBARS) assay was used to quantify the production of free radicals in the analysis of the effects of the extracts on oxidative stress. Lastly, gene expression of apoptosis-related genes was investigated using real-time polymerase chain reaction (RT-PCR). The results show that the crude extracts of A. betulina are selective towards the MCF-7 cells but not the MRC-5. The extracts resulted in low levels of ATP production in the cancer-treated cells. Low levels of extracellular LDH depicted in MCF-7 treated cells show that the cells are undergoing apoptosis, and not necrosis. This was further supported by the significant morphological and nuclear changes observed in cancer-treated cells with IC50 concentrations of methanol and ethyl acetate, i.e., cell shrinkage, spikes, degradation of the nuclear envelope, and nuclear degradation. Elevated levels of malondialdehyde (MDA) production in treated MCF-7 cells signify the stimulation of oxidative stress, meaning that the induced apoptotic ...