Background <p>Prostate cancer, a leading cause of cancer-related mortality in men, often develops resistance to conventional therapies, necessitating the discovery of novel therapeutic targets. Catalase plays a critical role in maintaining cellular redox homeostasis. Its inhibition disrupts oxidative balance, leading to oxidative stress and cell death, which positions catalase as a promising therapeutic target for cancer treatment. This study explores the anti-cancer effects and mechanisms of action of trichodermamide B in targeting catalase-induced apoptosis in prostate cancer cells.</p> Methods <p>The anti-prostate cancer activity of trichodermamide B was evaluated using multiple prostate cancer cell lines, including LNCaP, 22Rv1, PC-3, and DU145. Cytotoxicity was assessed through cell viability assays, while clonogenic assays were employed to measure the compound's ability to inhibit colony formation. The impact on 3D cell sphere growth was examined using LNCaP-3D models. Flow cytometry was utilized to analyze cell cycle arrest and apoptosis induction. Molecular docking studies were conducted to predict the interaction between trichodermamide B and catalase. The cell-free catalase enzymatic activity assay was employed to detect the inhibitory effect of trichodermamide B on catalase activity in vitro. Proteome profiler human apoptosis array kits and immunoblotting were used to validate the suppression of catalase expression. Additionally, the effects on androgen receptor (AR) activity and AR target genes (KLK3 and TMPRSS2) were investigated by western blot and qPCR. In vivo efficacy was confirmed using zebrafish and mouse xenograft tumor models.</p> Results <p>Trichodermamide B demonstrated potent cytotoxicity against all tested prostate cancer cell lines, with significant inhibition of clonal colony formation in 22Rv1 and PC-3 cells. It also impaired the growth of LNCaP-3D cell spheres, indicating its ability to target both 2D and 3D cancer models. Flow cytometry revealed that trichodermamide B induced cell cycle arrest and apoptosis. Molecular docking studies predicted a strong binding affinity between trichodermamide B and catalase. This was corroborated by experimental data showing that trichodermamide B inhibits the enzymatic activity of catalase in a dose-dependent manner in vitro. This inhibition led to disrupted redox homeostasis, accumulation of reactive oxygen species (ROS), and intense oxidative stress. In AR-dependent LNCaP cells, trichodermamide B suppressed AR activity and downregulated the expression of AR target genes. In vivo studies using zebrafish and mouse xenograft models further validated its anti-prostate cancer efficacy.</p> Conclusion <p>Trichodermamide B targets catalase while mediating crosstalk between the catalase axis and the AR signalling axis to induce oxidative stress and apoptosis in prostate cancer cells. Its efficacy in both in vitro and in vivo models underscore its viability as a novel drug candidate for prostate cancer treatment.</p>