Low-intensity ultrasound converts oleuropein into a potent inducer of intrinsic mitochondrial apoptosis in castration-resistant prostate cancer cells: An integrated in silico and in vitro study.

Metastatic castration-resistant prostate cancer (mCRPC) remains largely incurable because of its androgen receptor-independent phenotype and multidrug resistance. Oleuropein (OLE), the principal secoiridoid of the olive tree (Olea europaea L.), is a promising pro-apoptotic phytochemical, yet its poor membrane permeability curtails single-agent efficacy. We first mapped the affinity of OLE for the core regulators of the intrinsic apoptotic cascade (Bax, Bcl-2, caspase-3, caspase-9) by molecular docking, then tested whether low-intensity ultrasound (US; 1 MHz, 0.5 W/cm²) could unmask this potential in PC3 cells. Binding energies were computed with AutoDock Vina, and PC3 cells were assigned to six groups (Control, US, OLE50, OLE100, OLE50 +US, OLE100 +US). Viability (MTT), apoptosis (Annexin V-FITC/PI), mitochondrial membrane potential (JC-1), redox markers (MDA, GSH, SOD, CAT), cytokines (TNF-α, IL-6, IL-1β, IL-10) and the apoptotic proteins were quantified. Docking, validated by re-docking a co-crystallised reference ligand and by convergence testing across independent seeds, ranked the targets as caspase-9 (-7.27 kcal/mol) > caspase-3 (-7.23) > Bcl-2 (-6.50) > Bax (-5.93). The OLE IC50 was 147.92 µg/mL at 48 h and 116.32 µg/mL at 72 h, whereas US alone was non-cytotoxic. When sublethal OLE was combined with US, viability fell to ~26%, apoptosis reached ~74% and the mitochondrial membrane potential collapsed. The combination increased MDA, depleted GSH/SOD/CAT, elevated TNF-α/IL-6/IL-1β, suppressed IL-10, and raised the Bax/Bcl-2 ratio ~5.1-fold together with elevated cytochrome-c and cleaved caspase-9/-3. Oleuropein showed modest, closely spaced affinity for all four targets rather than a single high-affinity site, and the experimental activation pattern is discussed in relation to the in silico affinity ranking; taken together, the two layers are consistent with oleuropein engaging the intrinsic mitochondrial apoptotic machinery once ultrasound raises its intracellular availability, with ultrasound acting not merely as a permeability enhancer but as a co-driver of the pro-oxidant, pro-apoptotic shift. This non-invasive, externally controllable combination offers a rational synergistic strategy for mCRPC.