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

Abstract

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.