Efficacy of MDM2 and Epigenetic Inhibitors for Neuroblastoma Treatment

Abstract

Neuroblastoma (NB), a prevalent pediatric solid tumor originating from neural crest cells, remains a significant challenge in childhood cancer treatment, accounting for 15% of pediatric cancer-related deaths. This comprehensive study investigated the effects of two promising compounds, RG-7388 and CM-272, on neuroblastoma cells. RG-7388 is a potent MDM2 inhibitor, whereas CM-272 is a dual inhibitor targeting G9a and DNMT1. This study utilized two well-established neuroblastoma cell lines, SK-N-SH and IMR-32, to elucidate the molecular mechanisms underlying the anticancer properties of these compounds. Initial cytotoxicity studies were established for 24 hours. showed a decrease in the viability of SK-NSH and IMR-32 cells, in a time and concentration-dependent manner. Moreover, The experimental findings demonstrated that both RG-7388 and CM-272 effectively induced apoptosis in neuroblastoma cells, as evidenced by elevated levels of PARP and cleaved PARP with CM272 and activation of Caspase-3/7 confirmed through DEVD-amc substrate fluorescence analysis for both RG-7388 and CM-272. In addition, Immunofluorescence results revealed significant upregulation of p21 expression following both treatments. Western blot analysis confirmed distinct mechanisms of action: RG-7388 stabilized p53 by inhibiting MDM2, resulting in increased p53, MDM2, and p21 protein levels. At the same time, CM-272 showed no effect on p53 or MDM2 but significantly elevated p27 levels, suggesting its role in cell cycle regulation through epigenetic mechanisms via DNMT and G9a inhibition. RG-7388 and CM272 effectively downregulated anti-apoptotic proteins BCL-XL and XIAP, though neither affected autophagy markers Beclin-1, LC3-I, and LC3-II. qRT-PCR analysis revealed significant upregulation of CDKN1A (p21) mRNA and downregulation of PARP1 mRNA in treated cells, with CM-272 additionally reducing the expression of DNMT-associated genes. We conducted a gene expression analysis to elucidate further the molecular changes induced by these compounds. This comprehensive approach revealed significant alterations in the expression of cell cycle-related genes after CM- 272 treatment. These findings provide valuable insights into the distinct and complementary mechanisms by which RG-7388 and CM-272 exert their anticancer effects, advancing the understanding of targeted therapeutic strategies for high-risk neuroblastoma.