Stage-dependent antitumor effects of Icaritin in AOM/DSS-induced colon cancer: association with CPEB1 restoration and suppression of CSC-associated Wnt/β-Catenin and Notch Signaling pathways.

BACKGROUND: Cancer stem cells (CSCs) play a central role in colon cancer progression and are major contributors to metastasis, recurrence, and therapeutic resistance. CPEB1 (Cytoplasmic Polyadenylation Element Binding Protein 1) is a tumor suppressor, but its role in CSC-associated signaling during colon cancer progression remains insufficiently understood. The potential modulation of CPEB1 by Icaritin, a prenylated flavonoid, particularly in a stage-dependent context, has not been comprehensively investigated.

OBJECTIVE: To investigate the effect of Icaritin on CPEB1 expression, CSC-associated markers, apoptosis, inflammatory cytokines, and stemness-related signaling pathways during the early- and late-stage of colon cancer progression using an in vivo AOM/DSS-induced colon cancer model.

METHODS: In our study, colon cancer was induced in BALB/c mice using azoxymethane (AOM) and dextran sulfate sodium (DSS) administered in cycles (three cycles to model early-stage disease; six cycles to model advanced-stage disease). Tumor stage was confirmed by histopathological analysis, while western blotting was performed for CPEB1, CSC markers (CD133, ALDH1A1, Nanog, Sox2, CD44), apoptosis markers (Bcl-2, caspase-3, Bax), and key signaling pathway proteins (β-catenin, c-Myc, Notch1, Hes1). Serum cytokines IL-6, TNF-α, IL-1β, and IL-10 were measured using enzyme-linked immunosorbent assay (ELISA). Ki-67 immunohistochemical staining was performed to evaluate tumor proliferative activity.

RESULTS: Advanced-stage tumors exhibited significantly lower baseline CPEB1 expression and significantly higher CSC marker expression compared with early-stage tumors. Treatment with Icaritin was associated with a marked increase in CPEB1 expression in advanced-stage tumors, accompanied by a greater reduction in CSC marker expression and more pronounced reductions in the expression of β-catenin, c-Myc, Notch1, and Hes1. Apoptotic activation was more pronounced in advanced-stage models (as indicated by increased cleaved caspase-3 and Bax expression and decreased Bcl-2 levels). Cytokine modulation was also more substantial in advanced-stage disease, where IL-6, TNF-α, and IL-1β were markedly reduced, and IL-10 was significantly increased. Furthermore, Ki-67-positive proliferative activity was significantly elevated in advanced-stage tumors and was markedly reduced following Icaritin treatment.

CONCLUSION: Icaritin exhibits enhanced anti-inflammatory, pro-apoptotic, and CSC-associated molecular modulatory effects in advanced-stage colon cancer and is associated with upregulated CPEB1 expression and greater suppression of CSC-associated markers and reduced expression of proteins associated with stemness-related signaling pathways compared with early-stage disease. The observed association between increased CPEB1 expression and suppression of CSC-associated signaling pathways suggests a potential role of CPEB1 restoration in Icaritin-mediated antitumor responses; however, direct CPEB1-dependent regulatory mechanisms remain to be established. The observed reductions in CSC-associated markers represent molecular changes and do not establish direct inhibition of CSC self-renewal or functional activity. Further mechanistic and functional CSC studies are warranted to clarify the direct regulatory relationship between CPEB1 restoration and the antitumor effects of Icaritin in colorectal cancer progression.