PO.ET05.02 · 实验与分子治疗
药理浓度抗坏血酸通过ROS依赖的代谢脆弱性在MYCN扩增的神经母细胞瘤中发挥抗癌活性
Pharmacologic ascorbate exerts anticancer activity through ROS-dependent metabolic vulnerability in MYCN-amplified neuroblastoma
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：神经母细胞瘤（NB）是一种侵袭性儿童癌症，预后不良，尤其是伴有MYCN扩增（MNA）的患者。MYCN驱动的代谢可塑性使NB细胞即使在治疗压力下也能存活。药理浓度抗坏血酸（Asc）已成为一种潜在的低毒性抗癌药物，可产生过氧化氢（H₂O₂）并诱导氧化应激，选择性杀伤癌细胞。本研究探讨了MYCN扩增和非扩增NB细胞对抗坏血酸的ROS介导代谢脆弱性。方法：将患者来源的MYCN扩增（MNA）（COG-N-415）和非扩增（COG-N-618）神经母细胞瘤细胞用0-20 mM抗坏血酸处理24小时。使用CellTiter-Glo®测定法测量细胞活力。通过HPLC定量细胞内ATP水平。使用H₂O₂和过氧化氢酶评估ROS依赖性。在1-10 mM抗坏血酸处理后，通过Seahorse XF分析评估线粒体呼吸和糖酵解活性。结果：抗坏血酸在MNA和非MNA神经母细胞瘤细胞中均诱导剂量依赖性的细胞活力下降，在10 mM时约90%细胞死亡。COG-N-618（非MNA）细胞比COG-N-415（MNA）表现出更高的敏感性，在5 mM抗坏血酸时显示出显著的活力丧失。两种细胞在5 mM抗坏血酸处理后ATP水平均下降。过氧化氢酶挽救了抗坏血酸诱导的ATP耗竭，证实了H₂O₂介导的机制。Seahorse分析显示线粒体呼吸和糖酵解的渐进性抑制，COG-N-618细胞在1-10 mM时显示出剂量依赖性的氧化磷酸化（OCR）和糖酵解（ECAR）抑制。对于COG-N-415细胞（MNA），OCR和ECAR在1 mM抗坏血酸时均下降，但5 mM和10 mM未引起进一步下降，表明MYCN扩增细胞中存在适应机制。结论：药理浓度抗坏血酸在神经母细胞瘤细胞中诱导细胞毒性，并触发ROS依赖的代谢紊乱和MYCN下调。
查看英文原文 English abstract
Background: Neuroblastoma (NB) is an aggressive pediatric cancer with poor prognosis, particularly in patients with MYCN amplification (MNA). MYCN-driven metabolic plasticity allows NB cell survival even under therapeutic stress. Pharmacologic ascorbate (Asc) has emerged as a potential low-toxicity anticancer agent that generates hydrogen peroxide (H₂O₂) and induces oxidative stress, selectively killing cancer cells. This study investigated the ROS-mediated metabolic vulnerability of MYCN-amplified and non-amplified NB cells in response to ascorbate.
Method: Patient-derived MYCN-amplified (MNA) (COG-N-415) and non-amplified (COG-N-618) neuroblastoma cells were treated with 0-20 mM ascorbate for 24 hours. Cell viability was measured using the CellTiter-Glo® assay. Intracellular ATP levels were quantified by HPLC. H₂O₂ and catalase were used to assess ROS dependence. Mitochondrial respiration and glycolytic activity were assessed by Seahorse XF analysis after 1-10 mM ascorbate treatment.
Result: Ascorbate induced a dose-dependent decrease in cell viability in both MNA and non-MNA neuroblastoma cells, with approximately 90% cell death at 10 mM. COG-N-618 (non-MNA) cells exhibited greater sensitivity than COG-N-415 (MNA), showing significant viability loss at 5 mM ascorbate. ATP levels decreased in both cells upon 5 mM ascorbate treatment. Catalase rescued ascorbate-induced ATP depletion, confirming an H₂O₂-mediated mechanism. Seahorse analysis demonstrated progressive inhibition in mitochondrial respiration and glycolysis, with COG-N-618 cells showing dose dependent Oxidative Phosphorylation (OCR) and Glycolysis (ECAR) inhibition at 1-10 mM. For the COG-N-415 cells (MNA), both OCR and ECAR decreased at 1 mM of ascorbate, but 5 mM and 10 mM did not cause further decrease, indicating an adaptive mechanism in the MYCN-amplified cells.
Conclusion: Pharmacologic ascorbate induces cytotoxicity in neuroblastoma cells and triggers ROS-dependent metabolic disruption and MYCN downregulation.
利益披露 Disclosure
T. Farah, None..
Q. Chen, None.