Baicalein downregulates kinase-based and estrogen signaling pathways in breast cancer stem cells: an integrated...

Breast cancer stem cells (BCSCs) are one of the causes of drug resistance and disease recurrence due to their capacity for self-renewal and heterogeneity induction. A new BCSC-targeting agent has become a prospective approach to overcome resistance. Baicalein (5,6,7-trihydroxyflavone), a flavonoid extracted from Scutellaria baicalensis, has demonstrated anticancer activities in several models, including breast cancer. However, further elucidation of its effects on BCSCs is required. This study utilizes integrative bioinformatic approaches to identify the potential targets of baicalein in overcoming BCSC. In vitro experiments confirmed the top ten target genes recognized during a previous bioinformatic analysis of MCF-7 as a mammosphere for cytotoxicity and gene expression assays. We identified the potential baicalein target genes in BCSCs, which include CTNNB1, STAT3, BCL2, HIF1A, ESR1, TNF, CCND1, IL6, JUN, and MAPK3. Gene annotation and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis revealed a possible attenuation of the estrogen signaling pathway by baicalein and its involvement in cell cycle regulation. We successfully constructed a three-dimensional (3D) mammosphere culture and characterized it to possess higher levels of "stemness-associated" factors (OCT4 and SOX2) compared to two-dimensional (2D) cultures. Baicalein did not demonstrate any significant effects on cell viability in both 2D and 3D cultures, although a decline was observed in 2D cultures. qRT-PCR revealed that baicalein suppressed all hub genes. Furthermore, molecular docking confirmed the gene expression patterns and that baicalein had better binding affinity to CTNNB1, STAT3, TNF, JNK1, and mitogen-activated protein kinase (MAPK) than the respective native ligands. In addition, other proteins also interacted with baicalein, as reflected by the docking scores. Baicalein was identified to interact with ten potential targets through a bioinformatics study, although it did not exhibit cytotoxicity in 2D and 3D MCF-7 cultures. However, a downward trajectory was observed in the expression levels of hub genes related to kinase pathways, like Wnt/β catenin, PI3K/Akt, and MAPK, as well as inflammation-associated genes that correlate with BCSC survivability. One of the most prominent was the estrogen signaling pathway, which was supported by the molecular docking results. Future research directions included confirmation of baicalein's efficacy and toxicity through in vivo approaches, as well as to understand its efficacy as a combination chemotherapeutic agent.

The online version contains supplementary material available at 10.1007/s10616-026-00917-9.