Shoushi LiuXiaolan LiZhiping ChengKaili ZhaoYanting WangYuhe WeiYuanshuo WangJinjian LuXin Yang0000-0003-1377-7655Lim Yang MooiQinpei LuQiuju HuangHongwei Guo2026-10-192026-10-192026-0610.1016/j.phymed.2026.158143https://dspace-cris.utar.edu.my/handle/123456789/11792Background Prostate cancer (PCa) represents a leading cause of cancer-related mortality in men. Betulinic acid (BA) exhibits antitumor properties. However, its direct molecular target and mechanism in PCa remain unclear. Purpose To identify BA’s molecular target and elucidate its mechanism in inhibiting PCa progression. Study design In vitro and in vivo models were employed to evaluate BA’s antitumor effects and underlying mechanisms. Methods BA’s antitumor activity was assessed in PCa cell lines and xenograft models. Apoptosis was examined via Hoechst staining, flow cytometry, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Lysosomal membrane permeabilization (LMP) was examined via LysoTracker Red and acridine orange (AO) staining, validated by CA074-Me. Target identification utilized pull-down assays, drug affinity responsive target stability (DARTS), molecular docking, and microscale thermophoresis (MST). Clinical data analysis and siRNA knockdown confirmed DDX5?s role. Results BA significantly suppressed PCa growth in vitro and in vivo without toxicity. BA directly bound DDX5, inhibiting transcription factor EB (TFEB)-mediated lysosomal biogenesis and downregulating V-ATPase subunit H (ATP6V1H), essential for lysosomal acidification. This disruption induced LMP and lysosome-dependent cell death (LDCD) via apoptosis, confirmed by CA074-Me. DDX5 expression was decreased in PCa tissues. DDX5 knockdown abolished BA-induced LDCD and lysosomal dysfunction. Conclusion BA directly targets DDX5, inducing LDCD through the novel BA-DDX5-TFEB-ATP6V1H axis, exploiting lysosomal vulnerability in PCa. These findings underscore BA’s therapeutic potential and propose lysosomal membrane destabilization as a precision treatment strategy for PCa. © 2026 Elsevier GmbH.en-USBetulinic acidDEAD-box helicase 5 (DDX5)Lysosomal membrane permeabilization (LMP)Lysosome-dependent cell death (LDCD)Prostate cancer (PCa)AnimalsAntineoplastic AgentsPhytogenicApoptosisBetulinic AcidCell LineTumorDEAD-box RNA HelicasesHumansLysosomesMaleMiceInbred BALB CNudeMolecular Docking SimulationPentacyclic TriterpenesProstatic NeoplasmsTriterpenesXenograft Model Antitumor Assaysacridine orangeadenosine triphosphatasebetulic acidDEAD box helicase 5DEAD box proteinn (3 propylcarbamoyloxirane 2 carbonyl)isoleucylprolinepaclitaxelsmall interfering RNAtranscription factortranscription factor EBunclassified drugantineoplastic agentDdx5 proteinhumanpentacyclic triterpenetriterpeneanimal cellanimal experimentanimal modelanimal tissueantineoplastic activityapoptosisArticlebiogenesiscancer growthcell deathcell permeabilizationcontrolled studydata analysisdrug mechanismdrug protein bindingdrug targetingflow cytometrygene knockdownhuman cellin vitro studyin vivo studylysosomal membrane permeabilizationlysosomelysosome dependent cell deathmalemicroscale thermophoresismolecular dockingmousenonhumanprostate cancerprostate cancer cell lineprotein expressionprotein functionpull-down assaystainingTUNEL assayvalidation processxenograftanimalBagg albino mousedrug effectdrug screeningdrug therapygeneticsmetabolismnude mousepathologyprostate tumortumor cell linejournal-article