Epigenetic modulation by MDM2 and DNMT inhibitor promotes cell cycle arrest and programmed cell death in neuroblastoma cells

Neuroblastoma isa pediatricmalignancythat affects childrenmostly under the age of 5 yearsold. Thismalignancyoriginatesfrom neural crest cells (called neuroblasts) that can be found inthe adrenal glands, neck, chest, or pelvicareas. Although varioustreatments are availablefortreatingneuroblastoma, experimentaland literatureevidencesuggest that epigeneticmodificationscan triggercell cycle arrest and induceprogrammed cell death (PCD), such asapoptosis,providinga potentialtherapeuticavenue for achievinggreater success duringneuroblastoma treatments. Our study investigatedwhether the MDM2 and DNMT inhibitorscould block DNMTs, and concurrently upregulate TET and p21, to inducecell cycle arrest andapoptoticcell death inIMR-32 and SK-N-AS cells. To assess the effects of RG-7388, CM-272,and SGI-1027, the neuroblastoma cells were treated for 24 hrs, then qRT-PCR analyses wereconducted to evaluate the gene expressionlevels of DNMT-1, DNMT-3A, DNMT-3B, G9a,EZH2, TET-2, TET-3, MDM2, p53, p21, and PARP. In addition,the proteinexpressionlevels ofthe previouslylistedgenes were evaluated through western blot methods to quantifythe changesinepigenetic,cell cycle, and apoptosis-relatedbiomarkers.Interestingly, the qRT-PCR analysisshowed a downregulationof DNMT-1, DNMT-3A, DNMT-3B, G9a, and EZH2 levels intheCM-272 and SGI-1027 treated SK-N-AS cells compared to the control. In both IMR-32 andSK-N-AS cells, the western blot analysisrevealed DNMT-1 downregulation,cleavage of PARP,and upregulationof BAX followingthe drug treatments. So far, our findingsindicatethatRG-7388, CM-272, and SGI-1027 treatments effectivelydownregulate the expressionof DNMTs, whileupregulatingp21 levels inIMR-32 cells, leadingto cell cycle arrest andapoptosis.The abilityof these experimentalcompounds to inducecell cycle arrest and cell deathsuggests that further evaluationof these compounds could lead to the identificationof apromisingtherapeuticagent for treatingaggressiveneuroblastoma.(Thisprojectwas supported by the NationalPediatricCancer Foundation(NPCF) and The RoyalDames of Cancer Research Inc., Ft. Lauderdale, Florida)