Abstract

Retinoblastoma (RB), a malignant intraocular tumor, represents a critical public health challenge, particularly for pediatric populations, underscoring the urgent need for innovative therapeutic approaches and novel drug development. In this study, amentoflavone (AMF) was identified as a potential agent against RB cancer stem cells (RBCSCs). While CCK8 assays revealed moderate anti‐proliferative effects of AMF on RB cell lines, migration and invasion assays demonstrated its potent ability to suppress cancer cell motility. Tumorsphere formation assays further indicated that AMF significantly reduces RB cell stemness. qRT–PCR analysis showed that AMF downregulates expression of stem cell markers (CD44, CD133, Oct4, and Nanog) in a dose‐dependent manner. In vivo studies confirmed that AMF inhibits tumor metastasis and prolongs survival in RB mouse models. To elucidate its mechanism of action, RNA sequencing, molecular docking, and surface plasmon resonance (SPR) were employed. These analyses revealed that AMF directly targets smoothened (SMO) to disrupt the SHh signaling pathway, thereby suppressing RB stem cell (RBSC) self‐renewal and tumor progression. This work uncovers a previously unreported mechanism by which AMF hinders RB development and highlights its potential as a promising therapeutic candidate for this aggressive pediatric cancer.