一种萘酰亚胺-多胺缀合物对人肝癌HepG2细胞的杀伤作用

Inhibitory Effect of a Novel Naphthalimide-polyamine Conjugate on Proliferation of Human Hepatocellular Carcinoma HepG2 Cell Line

目的研究一种萘酰亚胺-多胺缀合物对人肝癌HepG2细胞的杀伤作用,并探讨其作用机制。方法以体外培养的人肝癌HepG2细胞为研究对象,噻唑蓝法检测细胞毒性,AO/EB/Hochest 33342染色观察细胞凋亡形态,DCFH-DA检测HepG2细胞内ROS变化,Rh123染色检测线粒体膜电位变化,免疫荧光法检测caspase-9,PARP-1表达,PI染色分析细胞周期变化,流式细胞术分析细胞凋亡率。结果噻唑蓝法显示,该萘酰亚胺-多胺缀合物呈剂量依赖性抑制HepG2细胞增殖,48 h的IC_(50)为17.28μmol·L^(-1),24 h的IC_(50)为29.71μmol·L^(-1),10μmol·L^(-1)该萘酰亚胺-多胺缀合物作用48 h后:细胞内活性氧簇、caspase-9荧光强度均显著升高(P<0.01,n=20),Rh123、PARP-1荧光强度显著减低(P<0.01,n=20);细胞增殖阻滞在S期;25μmol·L^(-1)萘酰亚胺-多胺缀合物作用细胞48 h后,高内涵活细胞成像系统可见细胞核固缩呈圆珠状凋亡特征,流式细胞术显示细胞早期凋亡率为33.13%,晚期凋亡率为30.99%。结论该萘酰亚胺-多胺缀合物对HepG2细胞具有较强的促凋亡作用,其机制与升高细胞内ROS水平,降低线粒体膜电位,进而活化caspase-9,通过线粒体途径诱导细胞凋亡有关,另外,该萘酰亚胺-多胺缀合物还通过下调PARP-1表达而发挥促凋亡作用。

OBJECTIVE To study the effect of a novel naphthalimide-polyamine conjugate on apoptosis of human hepatocellular carcinoma HepG2 cell line. METHODS MqT assay was used to evaluate the cell inhibition rate. The cellular morphous was detected with AO/EB/Hoeehst staining, the ROS was detected with DCFH-DA staining, the mitochondrial membrane potential（ Am. ）was detected with Rh123 staining,the expression of caspase-9 and PARP-1 was detected by immunofluoreseence method. The cell cycle was detected by HCS. Apoptosis of HepG2 cells was quantified by flow cytometry using Annexin V/PI stain. RESULTS Proliferation of HepG2 cells was inhibited significantly by the naphthalimide-polyamine conjugate in a dosage dependant manner. Under HCS, some HepG2 cells underwent a typical apoptotie morphologic change and the expression of ROS and caspase-9 was increased. The expression of PARP-1 and the mitochondfial membrane potential（ A^m ）was detected decreased. Flow cytometry indicates 64. 12% HepG2 cells were induced apoptosis after 48 h incubation with 25 Ixmol ~ L-I naphthalimide-polyamine conjugate. CONCLUSION The naphthalimide-polyamine conjugate could significantly induce the apoptosis of HepG2 cells by the mitochondrial pathway. The mechanism is coneemed with increasing ROS, decreasing the mitoehondrial membrane potential, upgrading caspase-9 and decreasing the expression of PARP-1.