Synergistic Induction of Caspase-8-Mediated Leukaemic Cell Death by Fisetin and Pinocembrin

Fisetin is a bioactive flavanol with reported anticancer activity, although its mechanisms in leukaemia and potential for combination therapy remain incompletely understood. This study investigated the cytotoxic and mechanistic effects of fisetin, alone and combined with pinocembrin, in human leukaemia cells. Cell viability, apoptosis, and cell cycle progression were assessed by flow cytometry; protein expression in Jurkat cells was assessed by Western blotting; and molecular docking was used to evaluate interactions with the Fas receptor. Drug interactions were quantified using ZIP synergy analysis, and cytotoxicity and clonogenic survival were evaluated using soft-agar colony formation assays in K562 cells. Fisetin significantly reduced cell viability and induced apoptosis, accompanied by caspase-8 cleavage, p62 accumulation, and CDK4 downregulation, consistent with activation of extrinsic apoptosis, impaired autophagic flux, and cell cycle inhibition in Jurkat cells. Docking analysis supported a potential interaction with the Fas receptor, which was confirmed using the Fas receptor antagonist Met-12. Co-treatment with pinocembrin enhanced fisetin-mediated cytotoxicity and produced synergistic effects, particularly in Jurkat cells (ZIP score > 10), while synergistic interactions at specific sub-IC₅₀ concentrations were also observed in K562 cells. Combination treatment further enhanced caspase-8 activation, reduced CDK4 expression in Jurkat cells, and significantly suppressed clonogenic survival in K562 cells compared with single-agent treatments. These findings suggest that fisetin promotes caspase-8-dependent apoptosis, potentially involving Fas-associated signalling, and highlight fisetin-pinocembrin combination therapy as a promising strategy for leukaemia treatment.