A small population of cells with stem cell-like properties in prostate cancer 
(PCa), called prostate cancer stem cells (PrCSCs) or prostate stemness-high 
cancer cells, displays highly tumorigenic and metastatic features and may be 
responsible for the therapy resistance. A small molecule, napabucasin (BBI608), 
recently have been identified with suppression of stemness-high cancer cells in 
a variety of cancers. However, the effects of napabucasin on PCa cells as well 
as PrCSCs isolated from PCa cells have not yet been defined. The effect of 
napabucasin on PCa cells in cell proliferation, colony formation, and cell 
migration in vitro were measured by MTS, colony formation assay, and Transwell, 
respectively. Flow cytometry was employed to evaluate cell cycle and cell 
apoptosis, and the effect on tumorigenesis in vivo was examined by tumor growth 
assays. Furthermore, the role of napabucasin on self-renewal and survival of 
PrCSCs was evaluated by their ability to grow spheres and cell viability assay, 
respectively. Western Blot and qRT-PCR were used to determine the effect of 
napabucasin on the expressions of stemness markers. Decrease in cell viability, 
colony formation, migration, and survival with cell cycle arrest, higher 
sensitivity to docetaxel in vitro, and repressed tumorigenesis in vivo was 
observed upon napabucasin treatment. More importantly, napabucasin can obviously 
inhibit spherogenesis and even kill PrCSCs in vitro. Downregulation of stemness 
markers was observed after PrCSCs were treated with napabucasin. This study 
demonstrates that napabucasin may be a novel approach in the treatment of 
advanced PCa, specifically for castration-resistant prostate cancer (CRPC).