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. Methods Western 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. Results The 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. Conclusion EPZ011989 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.

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