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 wer...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 mechanisms underlying these effects may involve intracellular DNA damage,disturbance of energy metabolism and mitochondrial dysfunction.展开更多
文摘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 mechanisms underlying these effects may involve intracellular DNA damage,disturbance of energy metabolism and mitochondrial dysfunction.
