Abstract
Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with high mortality rates and limited targeted therapies. TNBC stem cells (TNBCSCs) contribute to tumor aggressiveness, metastasis, and treatment resistance. Targeting TNBCSCs represents a promising therapeutic strategy for improving patient outcomes. In this study, we investigated the inhibitory effects of bufadienolides-bufalin, bufotalin, and cinobufotalin-on TNBCSC growth. Among them, bufalin exhibited the strongest antiproliferative activity. We further examined bufalin's impact on TNBCSC self-renewal, cell cycle regulation, apoptosis, and the Wnt/β-catenin signaling pathway using in vitro and in vivo models. Bufalin effectively suppressed TNBCSC self-renewal in in vitro tumorsphere assays and significantly reduced tumor growth in an in vivo HCC1937 TNBCSC xenograft chorioallantoic membrane (CAM) model. Bufalin induced G0/G1 phase cell cycle arrest by downregulating key regulatory proteins, including c-myc, cyclin D1, and CDK4. It also promoted intrinsic apoptosis through nuclear fragmentation, mitochondrial membrane potential reduction, and caspase activation. Additionally, bufalin downregulated key CSC markers, such as CD133, CD44, ALDH1A1, Nanog, Oct4, and Sox2. Notably, bufalin suppressed the Wnt/β-catenin signaling pathway by reducing β-catenin mRNA and protein expression, leading to the downregulation of EGFR, a downstream target of Wnt signaling. Our findings highlight bufalin as a potent chemotherapeutic agent capable of inhibiting TNBCSC growth by targeting stemness, proliferation, and apoptosis through Wnt/β-catenin signaling suppression. These results provide a strong rationale for further investigation of bufalin as a potential therapeutic strategy for TNBC treatment.