In vitro: Toxicity was evaluatedusing DU145 metastatic prostate tumor spheroids. Brachydin A decreasedcell viability in a time- and dose-dependent manner, starting from 40 μM at 48 h. Flow cytometry showed increased apoptosis and necrosis, with >61%apoptosis at concentrations ≥80 μM. High-content screening revealed mitochondrial depolarization. Westernblotting confirmed increased markers of DNA damage (cleaved-PARP,p-γ-H2AX), apoptosis (CASP3, CASP7, CASP8), and inflammation(NF-kB, TNF-α), suggesting brachydin A induces cell death byPARP-related. Here, H2AX is linked to prostate neoplasm.