Melphalan and Curcumin Induce Apoptosis in Retinoblastoma Cells Associated with STAT3 Signaling Modulation

Background/Objectives: Retinoblastoma treatment remains limited by therapeutic resistance and toxicity. While melphalan is a key chemotherapeutic agent, its efficacy is constrained by adverse effects. Curcumin has emerged as a potential adjunct owing to its capacity to regulate oxidative stress and oncogenic signaling pathways, including STAT3. This study aimed to assess the synergistic tumor-inhibitory effects of melphalan-curcumin combined treatment and to investigate the roles of ROS, apoptosis, and STAT3-associated signaling, including validation in a three-dimensional (3D) tumor spheroid model. Materials and Methods: Human retinoblastoma (WERI-Rb-1) and normal keratinocyte (HaCaT) cells were exposed to melphalan, curcumin and the combined treatment regimen. Cell viability was analyzed by MTT assay, and drug interactions were analyzed using the Chou-Talalay method. Migration was evaluated by scratch assay. Intracellular ROS levels were quantified using the DCFH-DA assay and confirmed by flow cytometry. Apoptosis was quantified by Annexin V/PI staining, and caspase activity was assessed colorimetrically and by immunocytochemistry. Cytokine levels were determined by ELISA, and gene expression profiling of STAT3 and apoptosis-associated genes were performed using qRT-PCR. Three-dimensional tumor spheroids were established to evaluate treatment responses in a physiologically relevant model. The contribution of ROS was further investigated using N-acetyl-L-cysteine (NAC) pretreatment. Results: The combination of melphalan and curcumin notably reduced WERI-Rb-1 cell viability in a synergistic manner (CI < 1) while exhibiting lower cytotoxicity in HaCaT cells, indicating selective antitumor activity. Co-treatment markedly inhibited cell migration and increased intracellular ROS levels. Cells pretreated with NAC significantly reduced ROS levels accumulation and moderately restored cellular viability, supporting a contributory role of oxidative stress. The combination treatment induced pronounced apoptosis, with increased early and late apoptotic cell populations, enhanced caspase-7 and caspase-9 activity, and elevated caspase-9 protein expression. These effects were associated with upregulation of pro-apoptotic genes (BAX, CASP3, CASP7, CASP9), downregulation of anti-apoptotic genes (BCL2, SURVIVIN), and reduction in STAT3 mRNA expression. In addition, the combination reduced pro-inflammatory cytokine levels. Importantly, these effects were recapitulated in 3D tumor spheroids, where the combination treatment reduced spheroid size and viability and induced structural disruption. NAC-mediated rescue experiments in 3D models further supported the notion that ROS contributes to, but is not solely responsible for, the observed effects. Conclusions: Overall, these results suggest that melphalan and curcumin exert synergistic and selective antitumor effects in retinoblastoma cells, associated with changes consistent with ROS-related effects, mitochondrial apoptotic processes, and STAT3-related transcriptional alterations rather than definitive pathway activation. The validation of these effects in a 3D tumor spheroid model provides additional support for the potential clinical significance of this combined treatment; however, additional protein-level and functional validation is required.