乙酰水杨酸增强多胺衍生物ANISpm抗肝癌作用及其机制研究

Acetylsalicylic acid strengthens the effects of ANISpm against hepatocellular carcinoma and its molecular mechanism

探讨乙酰水杨酸增强多胺衍生物ANISpm抗肝癌作用及其作用机制。采用MTT法检测细胞毒性;应用高内涵活细胞成像系统检测细胞内ANISpm含量的变化及对细胞凋亡和线粒体膜电位的影响;采用Western blotting方法检测细胞色素c,精脒/精胺乙酰转移酶,Caspase-3、-8、-9等蛋白表达的变化;应用高效液相色谱法检测细胞内多胺含量的变化。应用H22肿瘤移植模型评价对实体瘤生长的影响。实验结果显示,乙酰水杨酸体内外均具有增强ANISpm抗肝癌的作用,其机制为乙酰水杨酸通过上调肝癌细胞内精脒/精胺乙酰转移酶的活性,降低细胞内多胺含量从而增加了ANISpm的摄取,并通过ANISpm诱导肝癌细胞凋亡。该凋亡作用与ANISpm诱导细胞线粒体膜电位降低,促进细胞色素c释放以及活化Caspase-3、-8、-9等有关。

The objective of this study is to examine the effects of ANISpm,a novel polyamine naphthalimide conjugate,with acetylsalicylic acid against hepatocellular carcinoma in vivo and in vitro and elucidate its potential molecular mechanism.The proliferation inhibition was detected by MTT assay.Cell apoptosis,intracellular fluorescence intensity and mitochondrial membrane potential（MMP） were detected by high content screening（HCS） analysis.Polyamines content was analyzed by reverse-phase high performance liquid chromatography.Protein expression levels were quantified by Western blotting assay.The combination treatment strongly inhibited cell proliferation,induced cell apoptosis in HepG2 cells and H22 hepatoma cells,which was mediated by enhanced ANISpm uptake via up-regulation of spermidine/spermine N 1-acetyltransferase（SSAT） and depression of intracellular polyamine.Furthermore,this synergistic apoptosis was involved in mitochondria and death-receptor signal pathway.All these findings demonstrated that the combination treatment with acetylsalicylic acid and ANISpm resulted in synergistic antitumor effects on hepatoma cells.Thus,combination therapy with these agents may be useful as a potential template for the development of better chemotherapeutic strategy against hepatoma.