Cucurbitacin-B Triggers Fas/FasL-dependent Apoptosis via the Death Receptor Signalling Pathway in Human Breast Cancer Cell Line - MCF-7

Background: Globally, Breast Cancer (BC) ranks as the second most common cause of cancer-related death for women. The high mortality and recurrence rates contribute substantially to its fatality. Objectives: This research focused to investigate the anti-cancer potential of Cucurbitacin-B (Cu-B) against the human breast cancer cell line MCF-7, focusing on the Fas/ FasL-mediated death receptor signalling pathway. Materials and Methods: The anti-proliferative effect of Cu-B was assessed using the tetrazolium-based MTT assay, while morphological alterations were observed through phase-contrast microscopy. Apoptotic features were further examined using Acridine Orange/Ethidium Bromide (AO/EtBr) dual staining, and intracellular Reactive Oxygen Species (ROS) levels were quantified with DCFH-DA fluorescence assay. Nuclear alterations were investigated by DNA fragmentation analysis, and FasL gene expression was quantified by qPCR. Results: MTT assay revealed that Cu-B exhibited an IC₅₀ value of 74.836 µg/ mL. Morphological assessment demonstrated pronounced cellular shrinkage and structural disintegration. AO/EtBr staining confirmed an increased proportion of apoptotic cells in Cu-B- treated groups, which was corroborated by the elevated ROS of 7750±901.38 in the treatment group compared to 1333.33±125.83 in the control group, as indicated by DCFH-DA assay. DNA laddering further confirmed nuclear damage, and qPCR analysis showed significant upregulation of FasL expression in treated cells, supporting the activation of death receptor-mediated apoptosis. Conclusion: Cucurbitacin-B demonstrated potent anti-cancer activity against MCF-7 cells. The findings suggest that Cu-B induces apoptosis primarily through the extrinsic pathway by activating Fas/FasL-mediated death receptor signalling.