Betulinic Acid Induces Apoptosis and G2/M Cell Cycle Arrest in SH-SY5Y Neuroblastoma Cells via EAG1 Suppression

Aim: Neuroblastoma is the most common extracranial solid tumour in children and is associated with high mortality, particularly in advanced stages. Given the limitations of current treatment approaches, such as recurrence and drug resistance, novel agents with tumour-specific effects and minimal toxicity to normal cells are needed. Betulinic Acid (BA), a naturally occurring pentacyclic triterpene, has gained attention due to its selective antitumor properties. Material and Methods: This study investigated the cytotoxic and molecular effects of BA on SH-SY5Y neuroblastoma and epithelial cell lines, Vero. Cell viability was assessed using the MTT assay. Microscopic observations were conducted to evaluate morphological changes. Quantitative real-time PCR (qPCR) was performed to analyse the gene expression changes associated with apoptosis (Bax, Bcl-2, Caspase-3, p53, p21) and the cell cycle (CDK1, Cyclin B1, Ki-67, EAG1). Results: BA significantly reduced cell viability in both cell lines in a time- and dose-dependent manner, with SH-SY5Y cells showing greater sensitivity (IC₅₀=75 μM at 48 hours). Morphological changes such as cell shrinkage and reduced cell density were more pronounced in SH-SY5Y cells. In this cell line, BA treatment significantly upregulated pro-apoptotic genes (Bax, Caspase-3, p53, p21) and downregulated anti-apoptotic and proliferation-related genes (Bcl-2, CDK1, Cyclin B1, Ki-67, EAG1) (p