一氧化氮合酶对丝裂霉素C衍生物629细胞毒性的影响

Effect of inducible nitrogen monoxide synthase on tumour cells sensitivity to mitomycin C analogue 629 in vitro

目的评价一氧化氮合酶对丝裂霉素C(MMC)衍生物———5-氮丙啶基-3羟-基-1甲-基吲哚-4,7二-酮(629)的细胞毒性和乏氧选择性的影响。方法以人纤维肉瘤细胞株HT1080和其诱导型一氧化氮合酶(iNOS)基因转染的细胞克隆(iNOS9,iNOS12)为实验对象。四噻唑蓝(MTT)法检测MMC与其衍生物629的细胞毒性,比较乏氧和有氧条件下两种化合物半数抑制率(IC50)的差异;琼脂糖凝胶电泳和流式细胞术观察629作用后肿瘤细胞脱氧核糖核酸(DNA)的损伤情况,分析不同iNOS活性的肿瘤细胞对629敏感性的差异及原因。结果629的IC50较MMC低数百倍,是高细胞毒性的化合物;随着细胞中iNOS活性的增加,629对乏氧细胞的选择性损伤作用显著增强,DNA电泳显示629引起细胞坏死,造成细胞周期G2/M阻滞。结论MMC衍生物629是具有更高乏氧选择性的增敏化合物。

Aim To examine the effect of inducible nitrogen monoxide synthase （iNOS） on tumour cells chemosensitivity to mitomycin C （MMC） analogue 5-aziridinyl-3-hydroxyl-1-methylindole-4,7-dione （629） in vitro, and elucidate the possible role of iNOS in the metabolism of 629. Methods Human sarcoma cells （HT1080） and its iNOS gene transfected clones （iNOS9, iNOS12） were exposed to 629 at concentrations of 1 nmol·L^-1 -100μmol·L^-1. 3-[ 4,5-Dimethylthiazol-2-yl ] -2,5-diphenyltetrazolium bromide （MTT） assay, agarose electrophoresis and flow cytometric analysis were used to determine cell sensitivity, deoxyribonucleic acid （DNA） damage and the change of cell cycle in above process, respectively. All experiments were performed both in air and under hypoxia parallelly. Results 629 was more toxic than MMC, and enhanced cytotoxicity under hypoxia, which resulted in cell necrosis. Sixteen hours after treated with 629, HT1080 cells and related iNOS-transfected clone cells were obviously blocked in GJM phase. Conclusion iNOS plays dual roles in 629 metabolism, enhancing or decreasing the cytoxicity of 629 depending on the intracellular oxygen pressure Po2, which caused higher cytotoxicity to hypoxia cells of 629 with the increasing of iNOS activity.