Damage effect and mechanisms of cyclophosphamide to human neuroblastoma SH-SY5Y cells  

环磷酰胺对人神经母细胞瘤SH-SY5Y细胞的损伤效应及机制

作　　者：LI Jiajia WANG Jiao XIAO Wenyi WEI Donghui ZHANG Yongxiang JIANG Ning ZHOU Wenxia 李佳佳;王娇;肖文一;韦冬晖;张永祥;蒋宁;周文霞(军事医学研究院国家安全特需药品全国重点实验室,北京100850)

机构地区：[1]State Key Laboratory of National Security Specially Needed Medicines,Academy of Military Medical Sciences,Beijing 100850,China

出　　处：《中国药理学与毒理学杂志》2024年第8期561-574,共14页Chinese Journal of Pharmacology and Toxicology

基　　金：国家重点研发计划(2022YFC3500304)。

摘　　要：OBJECTIVE To investigate the damage effect and mechanisms of cyclophosphamide(CTX)and its active metabolite derivative 4-hydroperoxycyclophosphamide(4-HC)to human neuroblas⁃toma SH-SY5Y cells.METHODS SH-SY5Y cells were treated with CTX[0(cell control),0.01,0.1,1,5,10,20,40 and 80 mmol·L^(-1)]and 4-HC[0(cell control),0.01,0.1,1,5,10,20,40 and 80μmol·L^(-1)]for 48 h.Cell confluence and morphology were observed by the IncuCyte ZOOM system.Cell viability was assessed by CCK-8 assay.Lactate dehydrogenase(LDH)release was measured by LDH assay kit.SH-SY5Y cells were treated with CTX(0,1,5,10 and 20 mmol·L^(-1))and 4-HC(0,1,5,10 and 20μmol·L^(-1))for 48 h before cell proliferation was analyzed by 5-ethynyl-2′-deoxyuridine(EdU)staining assay.Immunofluorescence was employed to assess the levels of the DNA double-strand break markerγ-H2AX and to evaluate changes in mitochondrial membrane potential.SH-SY5Y cells were treated with CTX(0,1,5 and 10 mmol·L^(-1))and 4-HC(0,1,5 and 10μmol·L^(-1))for 48 h,and the alterations in glycolysis and oxidative phosphorylation levels were analyzed using the Seahorse XFe96 Analyzer.RESULTS Compared with the cell control group,cell confluence and cell viability were significantly reduced in the CTX and 4-HC groups(P<0.01),and the half-maximal inhibitory concentrations(IC50)for CTX and 4-HC were 4.44 mmol·L^(-1) and 4.78μmol·L^(-1),respectively.The release rate of LDH was signif⁃icantly increased while the percentage of EdU+cells was significantly reduced in the CTX and 4-HC groups(P<0.01).The percentage ofγ-H2AX+cells was significantly increased and mitochondrial membrane potential significantly decreased in the CTX and 4-HC group(P<0.05).Treatment with CTX and 4-HC resulted in reduced levels of maximum glycolytic capacity,glycolytic reserve,maximal respi⁃ration,and ATP production(P<0.05).CONCLUSION CTX and 4-HC exert significant cytotoxic effects on SH-SY5Y cells by disrupting cell membrane structure,impeding cell proliferation,and reducing cell viability.The mechanism目的探讨环磷酰胺(CTX)及其活性代谢衍生物4-氢过氧环磷酰胺(4-HC)对人神经母细胞瘤SH-SY5Y细胞的损伤效应及其机制。方法用CTX〔0(细胞对照),0.01,0.1,1,5,10,20,40和80 mmol·L^(-1)〕和4-HC〔0(细胞对照),0.01,0.1,1,5,10,20,40和80μmol·L^(-1)〕处理SH-SY5Y细胞48 h,应用IncuCyte ZOOM系统记录细胞形态变化和生长曲线;CCK-8法检测细胞活力;乳酸脱氢酶(LDH)试剂盒检测细胞LDH释放率。CTX(0,1,5,10和20 mmol·L^(-1))和4-HC(0,1,5,10和20μmol·L^(-1))处理SH-SY5Y细胞48 h,用5-乙炔基-2′-脱氧尿苷(EdU)染色法检测细胞增殖;免疫荧光法检测DNA双链断裂标志物γ-H2AX的含量和线粒体膜电位变化。CTX(0,1,5和10 mmol·L^(-1))和4-HC(0,1,5和10μmol·L^(-1))处理SH-SY5Y细胞48 h,SeaHorse XF检测细胞糖酵解及线粒体氧化磷酸化水平。结果与细胞对照组相比,CTX组和4-HC组的细胞融合率曲线逐渐下降;细胞活力显著下降(P<0.01),半数抑制浓度(IC50)分别为4.44 mmol·L^(-1)和4.78μmol·L^(-1);LDH释放率显著增加(P<0.01);EdU+细胞百分比显著降低(P<0.01);γ-H2AX+细胞百分比显著升高(P<0.05);线粒体膜电位显著降低(P<0.05);CTX和4-HC处理导致最大糖酵解能力、酵解储备、最大呼吸速率和ATP产生速率显著降低(P<0.05)。结论CTX和4-HC对SH-SY5Y细胞具有细胞毒性作用,可降低细胞活力,破坏细胞膜结构,抑制细胞增殖,其机制可能与导致细胞DNA损伤、能量代谢紊乱和线粒体功能障碍密切相关。

关 键 词：CYCLOPHOSPHAMIDE 4-hydroperoxycyclophosphamide NEUROTOXICITY energy metabolism 

分 类 号：R965.3[医药卫生—药理学] R979.1[医药卫生—药学]