Combined Modulation of Ceramide and S1P Pathways Induces Strong Cytotoxicity in SH-SY5Y Neuroblastoma Cells

Neuroblastoma is among the most aggressive pediatric cancers, and resistance to standard therapies highlights the need for alternative strategies targeting lipid metabolism and apoptosis-associated pathways. This study aimed to investigate the cytotoxic and apoptotic effects of fingolimod, ceranib-2, and carmofur, administered individually and in combination, in SH-SY5Y neuroblastoma cells, with particular emphasis on ceramide metabolism. Cytotoxicity was evaluated using the MTT assay, and morphological alterations were assessed by confocal microscopy. Colony-forming ability was examined using a soft agar assay, apoptosis-associated responses were evaluated by Annexin-V staining and caspase 3/7 activation analysis, and intracellular ceramide levels were determined using an ELISA-based method. The results showed that all three compounds reduced cell viability in a concentration-dependent manner and decreased clonogenic survival. The triple combination produced the most pronounced changes in apoptosis-associated parameters and showed marked inhibition of colony formation, although fingolimod alone exhibited the lowest IC₅₀ value and therefore remained the most potent treatment on an IC₅₀ basis. Confocal microscopy revealed prominent apoptosis-associated morphological alterations, including chromatin condensation, nuclear fragmentation, and cytoskeletal disruption, particularly in the triple-combination group. Annexin-V and caspase 3/7 analyses showed increased apoptotic cell populations, with the highest levels observed following triple-combination treatment. Intracellular ceramide levels were also significantly elevated, particularly in cells treated with ceranib-2 and the triple combination. Collectively, these findings indicate that simultaneous modulation of ceramide- and S1P-associated pathways influences apoptosis-associated responses, clonogenic growth, and intracellular ceramide levels in SH-SY5Y cells. However, because the study was conducted in a single neuroblastoma cell line and did not include comprehensive dose-by-time or formal combination-interaction analyses, the findings should be regarded as preliminary in vitro observations. Further validation in genetically distinct neuroblastoma models, together with systematic dose-response and time-response studies, will be required to determine the reproducibility, biological significance, and broader therapeutic relevance of this approach.