Methods To explore the anti-tumor effect of the zeste homolog enhancer 2 (EZH2) inhibitor EPZ011989 on retinoblastoma (RB) and its potential mechanism, and to evaluate whether it exerts an indirect neuroprotective effect on the optic nerve by regulating the epithelial-mesenchymal transition (EMT)-like process and the Wnt/β-catenin signaling pathway. MethodsWestern blotting (WB) was used to detect the expression of EZH2 protein in human retinal pigment epithelial cells (ARPE-19) and RB cells (Y-79, WERI-Rb-1). The half-maximal inhibitory concentration (IC50) of EPZ011989 on Y-79 and WERI-Rb-1 cells was determined using the Cell Counting Kit-8 (CCK-8). Y-79 and WERI-Rb-1 cells were treated with EPZ011989 at their respective IC50 concentrations and divided into untreated control groups (NC), dimethyl sulfoxide (DMSO) groups, and EPZ011989 treatment groups. Meanwhile, ARPE-19 cells were assigned to DMSO and EPZ011989 treatment groups. To verify the effect of EPZ011989 on the Wnt/β-catenin signaling pathway and its mechanism specificity, Y-79 and WERI-Rb-1 cells were respectively assigned to DMSO, EPZ011989 treatment, and EPZ011989 plus LiCl co-treatment groups. Cell proliferation, cell cycle, and apoptosis in different treatment groups were assessed by CCK-8 and flow cytometry, while WB was used to detect the expression of key proteins in the Wnt/β-catenin signaling pathway and EMT-related proteins.Animal experiment: 15 male C57BL/6J mice were randomly divided into 3 groups (n=5). A nude mouse RB subcutaneous transplantation tumor model was established by subcutaneous inoculation of Y-79 cells. The mice were orally administered with equal volume of DMSO (Control group), 100 mg/kg EPZ011989 (EPZ011989 low-dose group), and 300 mg/kg EPZ011989 (EPZ011989 high-dose group), twice a day for 4 weeks. Tumor volume and weight were monitored, and the expression levels of proliferation markers (Ki67) and EZH2 protein in the tumor-bearing tissues were detected by immunohistochemistry, and the expression levels of key proteins of the Wnt/β-catenin pathway in the tumor-bearing tissues were detected by WB. One-way analysis of variance was used for comparisons among multiple groups, and Tukey's post hoc test was used for pairwise comparisons between groups. ResultsThe expression levels of EZH2 protein in Y-79 and WERI-Rb-1 cells were significantly higher than those in ARPE-19 cells (P＜0.01). The IC50 values of EPZ011989 for Y-79 and WERI-Rb-1 cells were 1.851 μmol/L and 3.949 μmol/L, respectively. At these concentrations, EPZ011989 significantly inhibited the viability of both RB cell lines (P＜0.001) without significantly affecting the viability of ARPE-19 cells. Flow cytometry results showed that compared to the DMSO group, EPZ011989-treated Y-79 and WERI-Rb-1 cells exhibited consistent changes: the proportion of cells in the G0/G1 phase was significantly decreased (P＜0.001), while the proportions in the S and G2/M phases were significantly increased (P＜0.05); simultaneously, the apoptosis rates of both cell lines were significantly elevated (P＜0.001). Western blot analysis revealed that, compared to the DMSO group, EPZ011989 treatment significantly upregulated E-cadherin protein expression (P＜0.001) and downregulated N-cadherin and Vimentin expression (P＜0.001) in both RB cell lines, while also inhibiting the expression of key proteins in the Wnt/β-catenin pathway (Wnt1, β-catenin, c-MYC, Cyclin D1) (P＜0.01). In the rescue experiment with co-treatment of LiCl, compared with the EPZ011989 treatment group, the changes in cell cycle, increased apoptosis, inhibited Wnt/β-catenin pathway proteins (P＜0.001), and reversed EMT-like process-related proteins (down-regulation of E-cadherin, up-regulation of N-cadherin and Vimentin) caused by EPZ011989 were partially reversed in the EPZ011989+LiCl co-treatment group (P＜0.01). Tumor tissue detection showed that the tumor size in the high-dose EPZ011989 group was the smallest compared with the Control group and the low-dose EPZ011989 group. Compared with the Control group, the tumor volume and weight in the low-dose EPZ011989 group and the high-dose EPZ011989 group were significantly reduced (P < 0.001). Moreover, the reduction in tumor volume and weight in the high-dose EPZ011989 group was more significant (P＜0.001). The results of immunohistochemistry and WB assays showed that compared with the Control group, the expression of Ki67, EZH2, and key proteins of the Wnt/β-catenin pathway (Wnt1, β-catenin, c-MYC, Cyclin D1) in the low-dose EPZ011989 group and the high-dose EPZ011989 group were significantly decreased (P＜0.001), and the expression levels of these proteins in the high-dose group were the lowest. Conclusion: EPZ011989 inhibits the malignant progression of RB by suppressing the activation of the Wnt/β-catenin signaling pathway and may indirectly protect the function of the optic nerve by reducing the risk of optic nerve compression and regulating the EMT-like process and the Wnt/β-catenin signaling pathway. ConclusionEPZ011989 inhibits the malignant progression of RB by suppressing the activation of the Wnt/β-catenin signaling pathway, and may protect the optic nerve function indirectly by reducing the risk of optic nerve compression and regulating EMT-like processes and the Wnt/β-catenin signaling pathway.