Neuroblastoma, which originates from neural crest cells, is the most common extracranial solid tumor of childhood and is responsible for 15% of deaths. Despite standard therapies, survival rates in high-risk neuroblastoma cases are low and accelerate the search for new treatment strategies. Although p53 mutations are rare in neuroblastoma compared to other cancer types, they are observed in 15% of patients with neuroblastoma during recurrence. Evidence of p53 inactivation suggests that this may lead to chemotherapy resistance. MDM2 is a protein that regulates p53 and MDM2 inhibition can promote cell death by increasing p53 activation. Although RG7388, a potent and selective MDM2 inhibitor, has been shown to reduce survival in neuroblastoma cells, the underlying cell death mechanism has not been investigated yet. Etoposide, a part of neuroblastoma treatment, inhibits topoisomerase-II, which is involved in DNA replication and recombination and decreases cell survival. However, alternative combinations are needed due to the resistance mechanism developed by cancer cells against the apoptotic cell death pathway.

This study investigates the effect of combined RG7388 and etoposide treatment on cell survival in neuroblastoma cells. SHSY-5Y cells were seeded in 5x103 cells/well in 96 well plates. After the 24-hour incubation period, etoposide (A), RG7388 (B), and etoposide + RG7388 (C) were added to the growth medium for 24 or 48 hours. After this incubation period, the drug cytotoxicity screening was determined using an MTT colorimetric assay. All statistical analyses were performed using one-way analysis of variance (ANOVA) and followed by Tukey’s multiple comparison tests. A p-value less than 0.05 was considered to be significant.

IC50 values for etoposide were 64.2 and 4.4 µM for 24 and 48 hours, respectively. IC50 values of RG7388 were 40 and 7.6 µM for 24 and 48 hours, respectively. IC50 for combined treatment was determined as 6.09 µM for 48 hours. The combination index (CI) is above 1 at the combined IC50 dose, and antagonism is achieved, while at the combined dose that kills approximately 80% of the cells, the CI index is below 1 and synergism is achieved.

Combined treatment of MDM2 and topoisomerase-II inhibitors shows a synergistic effect in neuroblastoma and promotes cell survival. Elucidation of the type of cell death induced by these drugs may provide new insights into treating neuroblastoma. This study was supported Anadolu University within the scope of the project coded BGT-2023-2343.