Investigation of the anti-cancer potential of epoxyazadiradione in neuroblastoma: experimental assays and molecular analysis

Neuroblastoma, the most common childhood solid tumor, originates from primitive sympathetic nervous system cells. Epoxyazadiradione (EAD) is a limonoid derived from <i>Azadirachta indica</i>, belonging to the family Meliaceae. In this study, we isolated the EAD from <i>Azadirachta indica</i> seed and studied the anti-cancer potential against neuroblastoma. Herein, EAD demonstrated significant efficacy against neuroblastoma by suppressing cell proliferation, enhancing the rate of apoptosis and cycle arrest at the SubG₀ and G₂/M phases. EAD enhanced the pro-apoptotic Caspase 3 and Caspase 9 and inhibited the NF-kβ translocation in a dose-dependent manner. In order to identify the specific EAD target, a gel-free quantitative proteomics study on SH-SY5Y cells using Liquid Chromatography with tandem mass spectrometry was done in a dose-dependent manner, followed by detailed bioinformatics analysis to identify effects on protein. Proteomics data identified that Enolase1 and HSP90 were up-regulated in neuroblastoma. EAD inhibited the expression of Enolase1 and HSP90, validated by mRNA expression, immunoblotting, Enolase1 and HSP90 kit and flow-cytometry based bioassay. Molecular docking study, Molecular dynamic simulation, and along with molecular mechanics/Poisson-Boltzmann surface area analysis also suggested that EAD binds at the active site of the proteins and were stable throughout the 100 ns Molecular dynamic simulation study. Overall, this study suggested EAD exhibited anti-cancer activity against neuroblastoma by targeting Enolase1 and HSP90 pathways. Communicated by Ramaswamy H. Sarma

神经母细胞瘤（Neuroblastoma）是儿童最常见的实体瘤，起源于原始交感神经系统细胞。环氧印楝素二酮（Epoxyazadiradione，EAD）是一种柠檬苦素类化合物，源自楝科（Meliaceae）植物印楝（*Azadirachta indica*）。本研究从印楝种子中分离得到EAD，并探究其抗神经母细胞瘤活性。研究结果显示，EAD可通过抑制细胞增殖、促进细胞凋亡以及将细胞周期阻滞于SubG₀期与G₂/M期，发挥显著的抗神经母细胞瘤作用。EAD可呈剂量依赖性上调促凋亡蛋白半胱天冬酶3（Caspase 3）与半胱天冬酶9（Caspase 9）的表达，并抑制核因子κB（NF-κB）的核转位。为明确EAD的具体作用靶点，本研究以SH-SY5Y细胞为模型，采用液相色谱-串联质谱法（Liquid Chromatography with tandem mass spectrometry，LC-MS/MS）开展剂量依赖性的无胶定量蛋白质组学研究，并通过深入的生物信息学分析解析其对蛋白质表达的影响。蛋白质组学数据分析显示，烯醇化酶1（Enolase1，ENO1）与热休克蛋白90（HSP90）在神经母细胞瘤中呈高表达。EAD可下调烯醇化酶1与热休克蛋白90的表达，该结果经mRNA表达检测、免疫印迹实验、烯醇化酶1与热休克蛋白90检测试剂盒以及基于流式细胞术的生物实验验证。分子对接实验、分子动力学模拟（Molecular dynamic simulation）以及分子力学/泊松-玻尔兹曼表面积分析（Molecular mechanics/Poisson-Boltzmann surface area，MM-PBSA）结果均显示，EAD可结合于靶蛋白的活性位点，且在100 ns的分子动力学模拟过程中保持稳定。综上，本研究表明EAD可通过调控烯醇化酶1与热休克蛋白90相关通路，发挥抗神经母细胞瘤活性。本文由Ramaswamy H. Sarma转交刊发。