精胺氧化酶抑制剂的筛选及抗肿瘤活性评价

Screening and evaluation of antitumor activity of spermine oxidase inhibitors

目的筛选多胺代谢关键酶精胺氧化酶小分子抑制剂,并评价其对人肺癌A549细胞的精胺氧化酶活性抑制效应、抗肿瘤作用及分子机制。方法以精胺和精胺氧化酶复合物结构为基础,用计算机辅助药物设计技术,在化学数据库中虚拟筛选获得候选的小分子抑制剂。以A549细胞为肿瘤细胞模型,利用化学发光法、HPLC法分析小分子抑制剂对精胺氧化酶活性抑制及多胺含量的影响;采用MTT法、流式细胞术检测小分子抑制剂对细胞增值、细胞周期、诱导细胞凋亡的影响;在倒置荧光显微镜、透射电子显微镜下观察小分子抑制剂诱导细胞自噬小体的形成;Western blot法分析小分子抑制剂对细胞周期蛋白(Cyclin B1、p21、Cyclin D)、凋亡相关蛋白(Bax、Bcl-2、细胞色素C)和自噬相关蛋白(LC3、P62)表达的影响。结果筛选出有效抑制精胺氧化酶活性的新型小分子抑制剂SI-1338,对精胺氧化酶的半数抑制浓度为880μmol·L^(-1)。SI-1338可有效抑制A549细胞中精胺氧化酶活性,干扰多胺代谢,减少总多胺的含量;抑制A549细胞的增殖,周期蛋白Cyclin B1、p21、Cyclin D表达增加,细胞被阻滞在G_(0)/G_(1)期;A549细胞中Bax的表达增加,Bcl-2的表达减少,细胞色素C的释放增多,凋亡细胞的数量增加;LC3-Ⅱ片段增多,P62蛋白的表达量明显减少,诱导A549细胞中自噬小体形成增加。结论筛选获得新的小分子抑制剂SI-1338。该抑制剂对A549细胞具有高效抗肿瘤效应,其分子机制可能与抑制精胺氧化酶活性、干扰多胺代谢、诱导凋亡和自噬发生相关,有望作为以多胺代谢为靶点的抗肿瘤药物先导化合物。

OBJECTIVE To screen novel inhibitors of spermine oxidase(SMO),a key enzyme in polyamine metabolism,and investigate the antitumor effects of SMO inhibitors effect in human lung cancer A549 cells as well as their possible molecular mechanism.METHODS The combining pharmacophore modelling and molecular docking were carried out based on the complex structure of SMO-spermine for the virtual screening of SMO inhibitors.Using human lung cancer A549 cells as a tumor cell model,the chemiluminescence assay and HPLC were used to detemine enzyme activities of SMO and cellular polyamine content.MTT assay and flow cytometry(FCM)were used to analyze cell proliferation,cell cycle and apoptosis.Confocal laser scanning microscopy(LSCM)and fluorescence microscope were used to detect autophagy.Western blot was used to analyze the expression levels of cell cycle-related proteins(Cyclin B1,p21 and Cyclin D),apoptosis-related protein(Bax,Bcl-2 and cytochrome C)and autophagy-related protein(LC3,P62).RESULTS A novel inhibitor(SI-1338)was selected and showed inhibitory effect against SMO with IC_(50)value of 880μmol·L^(-1).SI-1338 could significantly inhibit the activity of SMO,interfere with polyamine metabolism and reduce the content of total polyamine in A549 cells.Treatment of A549 cells with SI-1338 inhibited proliferation,increased the expression levels of Cyclin B1,p21 and Cyclin D,caused G_(0)/G_(1)cell cycle arrest;increased the expression levels of Bax,cytochrome C and LC3-Ⅱfragment,decreased the expression levels of Bcl-2 and P62,induced apoptotic and autophagic cell death.CONCLUSION We have identified that SI-1338 functioned as a novel inhibitor of SMO.SI-1338 has the pharmacological activity of killing lung cancer A549 cells,and its mechanism may be related to the inhibition of SMO enzyme activity,interference of polyamine metabolism and induction of apoptosis and autophagy.SI-1338 can be used as a lead compound of antitumor drug with polyamine metabolism as target for cancer therapy.