Abstract
Pancreatic cancer remains a formidable global health challenge, characterised by low survival rates, with 510,992 cases and 467,409 deaths reported in 2022. Current cancer treatments, such as chemotherapy, radiation therapy, immunotherapy and surgery, are expensive and limited, primarily by their inability to differentiate between cancerous and non-cancerous cells. Consequently, investigating alternative anticancer treatments, such as medicinal plants, is crucial. This study aimed to investigate the potential anticancer properties of a crude methanolic extract and a fraction from Carpobrotus edulis to target the apoptotic machinery in pancreatic cancer cells (MIA PaCa2) to suppress their growth. Multiple assays, including Alamar blue, Adenosine triphosphate (ATP), Hoechst staining, Caspase 3/7 assay, Deoxyribonucleic acid (DNA) fragmentation, and reverse transcription quantitative polymerase chain reaction (RT-qPCR), were conducted to assess apoptosis induction. A fraction was obtained via silica-gel column chromatography and thin-layer chromatography (TLC), with Gas Chromatography-Mass Spectrometry (GC-MS) identifying two major compounds within the fraction for further analysis. The crude extract significantly reduced MIA PaCa2 cell viability at a minimum inhibitory concentration (IC50) of 85.44 μM, demonstrating high selectivity with a selectivity index (SI) of 3.65 towards the pancreatic cancer cell line. The fraction also showed significant cell viability reduction at an IC50 of 120 μM for MIA PaCa2 cells. The crude methanol extract and its fraction did not affect the viability of the non-cancerous medical research cell strain 5 (MRC-5) cell line. RT-qPCR confirmed that the crude methanol extract activated the extrinsic tumour necrotic factor-mediated (TNF-mediated) apoptosis pathway, evidenced by a significant increase in TNF expression and caspase 3/7 activity, leading to DNA and nuclear fragmentation, chromatin condensation, and the formation of apoptotic bodies. The loss of cell confluence, supported by non-significant (p > 0,05) downregulation of Signal Transducer and Activator of Transcription 3 (STAT3), further suggested inhibited cell growth. While some changes were observed in the expression of mutated protein 53(p53), BCL2-associated killer 1(Bak1), and Fas cell surface receptor (Fas) for both the crude extract and its fraction, these were not statistically significant (p > 0,05). Protein-ligand docking studies of the identified 7,9-Ditert-butyl-oxaspiro [4.5] deca-6,9-diene-2,8-dione and 1-octadecanol proved the compounds within the fraction as having high
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affinity for the selected ligands: TNF-receptor (TNFR), Fas-associated Death Domain (FADD), p53, Bak1, and STAT3. The compound 7,9-Ditert-butyl-oxaspiro [4.5] deca-6,9-diene-2,8-dione exhibited the most favourable binding energy, indicating its superior potential as a drug candidate. In conclusion, both the crude extract and its fraction demonstrated promising anticancer properties. The crude extract effectively activated the TNF-mediated apoptosis pathway, while the fraction showed encouraging in vitro and in silico anticancer activities. Future research will focus on further purification and investigation of the most potent compound within the fraction.